Methods for chemotaxis / redox driven separation of x and y chromosome bearing sperm and their insemination in gender specific menstrual cycles

ABSTRACT

The present invention provides a method for determining the gender of a fetus by assaying the sex hormones, evaluating the overall reducing/oxidizing (redox) activity, and/or evaluating the radical scavenging capacity of the maternal urine or other body fluid. The method can be used to determine fetal gender at any time point during pregnancy. The methods of the present invention also provide for a means for pre-conception offspring gender planning by assaying the sex hormones in urine samples, evaluating the overall reducing/oxidizing (redox) activity, and/or evaluating the radical scavenging capacity of the urine or other body fluid from a non-pregnant female. In some embodiments, the methods described herein are used to sort sperm based on an overall reducing/oxidizing environment of follicular fluids, which can then be used for intrauterine insemination.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part application of U.S. patentapplication Ser. No. 14/312,534, filed on Jun. 23, 2014; which is acontinuation application of U.S. patent application Ser. No. 12/884,079,filed on Sep. 16, 2010, now issued as U.S. Pat. No. 8,759,109; which isa continuation-in-part of U.S. patent application Ser. No. 12/401,723,filed on Mar. 11, 2009, now issued as U.S. Pat. No. 9,057,720; whichclaims the benefit under 35 U.S.C. § 119(e) of U.S. ProvisionalApplication No. 61/069,008, filed on Mar. 11, 2008. The disclosure ofeach of the above applications is incorporated by reference herein intheir entirety.

This application also claims the priority benefit under 35 U.S.C. §119(e) of U.S. Provisional Application No. 62/519,797, filed Jun. 14,2017, the disclosure of which is herein incorporated by reference in itsentirety.

TECHNICAL FIELD

The present invention relates to methods for determining the gender ofan unborn child by assaying the sex hormones, evaluating the overallreducing/oxidizing activity, and/or evaluating radical scavengingcapacity of the maternal urine. The present invention also relates tomethods for pre-conception baby gender planning by assaying the sexhormones, evaluating the overall reducing/oxidizing activity, and/orevaluating radical scavenging capacity of a non-pregnant female's urineprior to conception. The present invention also relates to a method ofseparating X and Y chromosome containing sperm, without the use of anyphysical, electric, electronic, magnetic, or centrifugal force,mimicking natural sperm selection in the uterus based on the forces ofchemotaxis and redox properties offered by internal sex hormonesreleased by follicular fluid. Such methods described herein may be usedin intrauterine insemination (IUI) or in-vitro fertilization (IVF)techniques to increase their success rates.

BACKGROUND

There is a great interest in accurately determining the gender of anunborn child as early as possible. Available techniques are typicallyperformed at late stages of a pregnancy. Ultrasound can be used todetermine fetal gender after the 18th week of pregnancy. Polymerasechain reaction (PCR) amplification of Y-specific DNA sequences requiresa sample of maternal blood, as well as expensive equipment. PCR can becarried out as early as the 6th week of gestation with an accuracy ofonly around 80%. Lagona et al. Multiple testing in fetal genderdetermination from maternal blood by polymerase chain reaction. Hum.Genet. 102, 6:1 (1998) Amniocentesis is performed after the 18th week ofpregnancy; however, it carries a risk of miscarriage due to itsinvasiveness. Chorionic Villi Sampling (CVS), conducted between the 10thand 13th week of pregnancy, can also provide accurate information, butagain, the procedure is invasive and requires costly equipment.

Alternatively, it is possible to determine the gender of an unborn childby assaying sex hormones. Sex hormones are steroids that play importantroles in both normal growth and development. In addition, sex hormonesinfluence the development of sex organs and maintenance of secondary sexcharacteristics in mammals. Testosterone, the principal male sexhormone, is primarily secreted by the testes of males and, to a muchless extent, the ovaries of females. On average, an adult man producesabout forty to sixty times more testosterone than an adult womanSimilarly, estrogen functions as the primary female sex hormone and isusually present at significantly higher levels in women than men. Theurine from a mother carrying a male fetus is richer in testosterone whencompared with the urine from a mother carrying a female fetus. Thisdifference is probably due to the contribution of sex hormones secretedby fetal testes. Jost A. A new Look at the Mechanisms Controlling SexDifferentiation in Mammals. John Hopkins Med. J. 130: 38 (1972).Approaches based on a pregnant woman's individual sex hormone levelshave been explored to determine fetal gender; however, thus far, theyhave not resulted in the development of any statistically reliablemethods. For example, in one study, antibodies against testosterone wereused in a radio-immunoassay to determine the testosterone levels inpregnant mothers' urine. The accuracy of this study with respect todetermining fetal gender was low. Loewit et al. Determination of fetalsex from maternal testosterone excretion in early pregnancy. Dtsch. Med.Wschr. 99: 1656 (1974).

Urine from pregnant women has been used in various attempts to developsimple and non-invasive tests to determine fetal gender. Most of theseprocedures exploit simple characteristics of maternal urine such as itspH or the ability to form complexes with aluminum compounds.Consequently, these tests are usually associated with comparatively poorreliability. For example, U.S. Pat. No. 6,420,182 discloses a method forfetal gender detection by assaying the pH of maternal urine after the12th to 14th week of pregnancy. The test is based on the hypothesis thatwomen bearing female fetuses have acidic urine, but the accuracy of thismethod is only about 65%. In another assay, a colorimetric test on urinefrom pregnant women may be performed after the 20th week of pregnancy.U.S. Pat. No. 4,840,914. The accuracy of this test in determining fetalgender was similarly low, only about 60%.

The applicant has noted that assaying multiple sex hormones, evaluatingthe overall reducing/oxidizing activity, and/or evaluating radicalscavenging capacity from urine sample collected near ovulation beforepregnancy has significant similarity and correlation with the genderoutcome after pregnancy. This suggests that biochemical environment inthe uterus existing at the time of conception may be locked andmaintained throughout pregnancy. Therefore, evaluation of theseparameters before pregnancy would provide reliable methods forpre-conception baby gender planning, as well as provide reliable methodsfor determining fetal gender after pregnancy. In other words, inaddition to X or Y chromosome bearing sperms, the gender of a baby isalso influenced by the biochemical environment of the uterus at the timeof conception.

Depending upon the processing and assay methods, the hormones which cancontribute to the reducing or oxidizing activity of a urine sample areprogesterone, testosterone, human chorionic gonadotropin (hCG), andestrogens. For example, estrogens can act as antioxidants, andcontribute to the radical scavenging capacity of the urine. The primaryhormones directly linked to fetal gender are sex hormones, namely,testosterone and estrogens. The overall reducing/oxidizing activityand/or radical scavenging capacity of the urine sample from a pregnantmother carrying a male fetus is different from that of the urine samplefrom a pregnant mother carrying a female fetus, thereby allowing fordetermination of fetal gender.

There is a continuing need to develop a simple, non-invasive andreliable method for determining the gender of an unborn child as earlyas possible after conception. There is also a need for pre-conceptionbaby gender planning using a simple, non-invasive and reliabletechnique. Because human and other mammals have similar reproductivebiology, these methods may also be of great commercial value to animalbreeders.

Further, such redox based pre-conception evaluation of gender specificcompatibility of an ovum, post-conception gender determination andrelatively more natural sperm sorting techniques may be useful inraising the success rates of artificial fertilization techniques (ART).Current sperm sorting/separation techniques use one or more externalforces, including multi-gravitational force, chemical gradient, orelectronic pulse etc. Such forces may damage sperm physically orbiochemically including DNA damage resulting in less successful ART. Forexample, Fluorescence Activated Cell Sorting (FACS) uses a cytometer incombination with DNA binding dyes and fluorescence detection. It hasbeen used for detecting minute differences in fluorescence signalsoriginating from a 3.8% difference in total DNA content between X and Ychromosome bearing bovine spermatozoa (G. E. Seidel ̂ D. Garner,Reproduction, 2002, 124,733-743). When the difference in DNA content isdetected, an applied electric field sorts the droplet containing thespermatozoa into X and Y chromosome bearing bins. The system can reachup to 25,000 individual spermatozoa per second and is useful inagricultural industries. However, it is known that high shear stress andhigh voltage cause extensive DNA fragmentation (D. H. Triyoso & T. A.Good, J. Physiol., 1999, 515, 355-365. and M. Stacey, et al, Mutat, Res,Genet, Toxicol, Environ, Mutagen., 2003, 542, 65-75).

Furthermore, current sperm sorting is imperfect, that is, the separationresults in a partially enriched fraction containing a 70/30% mixture ofX/Y chromosome containing sperm, potentially resulting in the gender ofthe fetus being other than the parents expected/wanted.

Thus, there is an urgent need of a technique of sorting and effectivelyseparating X and Y chromosome containing sperm, which does not involveany exposure to external physical or biochemical stress. There is also aneed to use this approach on natural gender specific conception, eitherat home or by using intrauterine insemination or in-vitro fertilization.If the technology described herein is used in proper gender specificmenstrual cycles, couples, fertile as well as less fertile, can conceivethe babies of desired gender, with much higher success rate thanexisting rates of 10-35% offered by current ARTs.

SUMMARY

It is an object of the present invention to provide a method ofdetermining the gender of an unborn child comprising the steps of: (a)measuring levels of testosterone (T), estrogens (E), progesterone (P)and gonadotropins (G) in a body fluid from a pregnant female; (b)calculating [E−(T+P+G)] as the difference between the level of estrogens(E) and the total levels of testosterone (T), progesterone (P) andgonadotropins (G); and (c) determining the gender of the unborn child tobe male if the [E−(T+P+G)] value from step (b) ranges from about 300 toabout 1,500, or to be female if the [E−(T+P+G)] value from step (b)ranges from about −10 to about −800. Various steps of the method may beconducted by a human or a machine.

The measurement in step (a) may further comprise a step of contactingthe body fluid with testosterone-specific, estrogens-specific,progesterone-specific or gonadotropins-specific antibodies. The level ofthe sex hormone associated with the antibodies may be determined byELISA, radioimmunoassay, Western blot, immunoprecipitation, or surfaceplasmon resonance (SPR) immunoassay. The antibodies can be attached to asolid phase, such as a surface of a cover slip, slide, tube, microtiterwell, sheet, chip, reaction tray, strip, membrane, film, fiber, plate,bottle or box. The solid phase may also be a particle, such as a silica,gold, nickel, polystyrene or latex particle. The particle may be ananoparticle having a diameter between about 0.1 nm and about 100 nm. Inone embodiment, the particle comprises a fluorescent compound positionedwithin the particle; the fluorescent compound of the particle attachedto testosterone-specific antibodies, the fluorescent compound of theparticle attached to estrogens-specific antibodies, the fluorescentcompound of the particle attached to progesterone-specific antibodies,and the fluorescent compound of the particle attached togonadotropins-specific antibodies are all different.

The levels of testosterone, estrogens, progesterone and gonadotropinsmay be measured by mass spectrometry, HPLC or multiplex technology.

The method may further comprise a step of attaching the testosterone,estrogens, progesterone and gonadotropins to a solid surface before step(a), such as a surface of a cover slip, slide, tube, microtiter well,sheet, chip, reaction tray, strip, membrane, film, fiber, plate, bottleor box.

The body fluid can be urine, tears, saliva, sweat, blood, plasma, serum,cerebrospinal fluid and amniotic fluid. The body fluid may be obtainedduring a period ranging from about four days before menstrual due dateto about 40 weeks of pregnancy, or during a period ranging from about 4weeks to about 15 weeks of pregnancy.

The present invention also provides a method of determining the genderof an unborn child comprising the steps of: (a) measuring levels oftestosterone (T), estrogens (E), progesterone (P) and gonadotropins (G)in a body fluid from a pregnant female; (b) calculating (E+T)/(P+G) asthe ratio of the total levels of estrogens (E) and testosterone (T) tothe total levels of progesterone (P) and gonadotropins (G); and (c)determining the gender of the unborn child to be male if the (E+T)/(P+G)ratio from step (b) is greater than about 1.4, or to be female if the(E+T)/(P+G) ratio from step (b) is less than about 1.2.

Further provided in the present invention is a method of determining thegender of an unborn child comprising the steps of: (a) performinghydroxyl radical antioxidant capacity (HORAC) assay on a body fluid froma pregnant female; (b) calculating the HORAC value; and (c) determiningthe gender of the unborn child to be male if the HORAC value from step(b) is less than about 5000, or to be female if the value from step (b)is greater than about 6000.

Also provided in the present invention is a method of determining thegender of an unborn child comprising the steps of: (a) performinghydroxyl radical antioxidant capacity (HORAC) assay on a body fluid froma pregnant female; (b) measuring levels of testosterone (T) andestrogens (E) in the body fluid, wherein step (b) can be performedbefore or after step (a); (c) calculating the value of HORAC/(E/T),wherein E/T is the ratio of the level of testosterone to the level ofestrogens, and wherein the value of HORAC/(E/T) is the ratio of theHORAC value to the E/T ratio; and, (d) determining the gender of theunborn child to be male if the value of HORAC/(E/T) from step (c) isless than about 150, or to be female if the value from step (c) isgreater than about 200.

The present invention provides for a method of determining the gender ofan unborn child comprising the steps of: (a) performing hydroxyl radicalantioxidant capacity (HORAC) assay on a body fluid from a pregnantfemale; (b) measuring levels of testosterone (T), estrogens (E),progesterone (P) and gonadotropins (G) in the body fluid, wherein step(b) can be performed before or after step (a); (c) calculating the valueof HORAC4E−(T+P+G)1, wherein [E−(T+P+G)] is the difference between thelevel of estrogens (E) and the total levels of testosterone (T),progesterone (P) and gonadotropins (G), and wherein the value ofHORAC/[E−(T+P+G)] is the ratio of the HORAC value to the [E−(T+P+G)]value; (d) comparing the HORAC/[E−(T+P+G)] value from step (c) with atleast one standard; and (e) determining the gender of the unborn child.

The present invention further provides a method of determining thegender of an unborn child comprising the steps of: (a) performinghydroxyl radical antioxidant capacity (HORAC) assay on a body fluid froma pregnant female; (b) measuring levels of testosterone (T), estrogens(E), progesterone (P) and gonadotropins (G) in the body fluid, whereinstep (b) can be performed before or after step (a); (c) calculating thevalue of HORAC/[E+T)/(P+G)] wherein [(E+T)/(P+G)] is the ratio of thetotal levels of estrogens (E) and testosterone (T) to the total levelsof progesterone (P) and gonadotropins (G), and wherein the value ofHORAC/[(E+T)(P+G)] is the ratio of the HORAC value to the [(E+T)/(P+G)]value; (d) comparing the HORAC/[E+T)/(P+G)] value from step (c) with atleast one standard; and, (e) determining the gender of the unborn child.

Also provided is a method of determining the gender of an unborn childcomprising the steps of: (a) contacting a body fluid from a pregnantfemale with free radicals; (b) measuring free radical scavengingactivity of the body fluid; (c) comparing the free radical scavengingactivity of the body fluid with at least one standard; and (d)determining the gender of the unborn child. The free radicals may beDPPH (1,1-Diphenyl-2-Picrythydrazyl) radical or GV(2,6-di-tert-butyl-α-(3,5-di-tert-butyl-oxo-2,5-cyclohexadien-1-ylidene)-p-tolyloxyradical. The free radicals may also be hydroxyl radicals or superoxideradicals. The hydroxyl radicals can be generated either internally orexternally using heat, irradiation, photolysis, ultrasound, electronparamagnetic resonance, electrolysis, chemical, electrochemical orenzymatic means. The hydroxyl radicals may be generated by Fentonreaction using iron ions, copper ions or titanium dioxide as catalysts.

In one embodiment, the gender of the unborn child is determined to befemale if decolorization of DPPH occurs within about 1 minute; thegender of the unborn child is determined to be male if decolorization ofDPPH occurs after more than about 1 hour. In another embodiment, thegender of the unborn child is determined to be male if decolorization ofDPPH occurs within about 15 minutes; the gender of the unborn child isdetermined to be female if decolorization of DPPH occurs from about 1hour to about 24 hours. DPPH may be in a solid form, in a solution, orattached to a solid surface.

The free radical scavenging activity can be measured by methylene bluethat is in a solid form, in a solution or attached to a solid surface.In one embodiment, the gender of an unborn child is determined to befemale if there is substantially no decolorization of methylene blue;the gender of an unborn child is determined to be male if there isdecolorization of methylene blue within about 1 hour.

The present invention further provides for a method of determining thegender of an unborn child comprising the steps of: (a) contacting a bodyfluid from a pregnant female with a solid surface having antibodiesspecific to estrogens, antibodies specific to testosterone, antibodiesspecific to progesterone and antibodies specific to hCG-beta; (b)contacting the solid surface from step (a) with at least one redoxindicator; (c) measuring redox activity of the solid surface; (d)comparing the redox activity of the solid surface with at least onestandard; and (e) determining the gender of the unborn child. The redoxactivity may be measured by Ferric reducing/antioxidant power (FRAP)assay or Ferric reducing/antioxidant power using ferrictripyridyltriazine (FRAP-TPTZ) assay.

The present invention also provides for a method of determining genderspecific compatibility of an ovum released in a menstrual cycle forpre-conception baby gender planning comprising the steps of: (a)measuring levels of testosterone (T), estrogens (E), progesterone (P)and gonadotropins (G) in a body fluid from a non-pregnant female; (b)calculating [E−(T+P+G)] as the difference between the level of estrogens(E) and the total levels of testosterone (T), progesterone (P) andgonadotropins (G); and (c) determining the gender specific compatibilityof the ovum released in the menstrual cycle to be male if the[E−(T+P+G)] value from step (b) ranges from about 300 to about 1,500, orto be female if the [E−(T+P+G)] value from step (b) ranges from about−10 to about −800.

Further provided in the present invention is a method of determininggender specific compatibility of an ovum released in a menstrual cyclefor pre-conception baby gender planning comprising the steps of: (a)measuring levels of testosterone (T), estrogens (E), progesterone (P)and gonadotropins (G) in a body fluid from a non-pregnant female; (b)calculating (E+T)/(P+G) as the ratio of the total levels of estrogens(E) and testosterone (T) to the total levels of progesterone (P) andgonadotropins (G); and (c) determining the gender specific compatibilityof the ovum released in the menstrual cycle to be male if the(E+T)/(P+G) ratio from step (b) is greater than about 1.4, or to befemale if the (E+T)/(P+G) ratio from step (b) is less than about 1.2.

Also provided in the present invention is a method of determining genderspecific compatibility of an ovum released in a menstrual cycle forpre-conception baby gender planning comprising the steps of: (a)performing hydroxyl radical antioxidant capacity (HORAC) assay on a bodyfluid from a non-pregnant female; (b) calculating the HORAC value; and(c) determining the gender specific compatibility of the ovum releasedin the menstrual cycle to be male if the HORAC value from step (b) isless than about 5000, or to be female if the value from step (b) isgreater than about 6000.

The present invention provides for a method of determining genderspecific compatibility of an ovum released in a menstrual cycle forpre-conception baby gender planning comprising the steps of: (a)performing hydroxyl radical antioxidant capacity (HORAC) assay on a bodyfluid from a non-pregnant female; (b) measuring levels of testosterone(T) and estrogens (E) in the body fluid, wherein step (b) can beperformed before or after step (a); (c) calculating the value ofHORAC/(E/T), wherein E/T is the ratio of the level of testosterone tothe level of estrogens, and wherein the value of HORAC/(E/T) is theratio of the HORAC value to the E/T ratio; and (d) determining thegender specific compatibility of the ovum released in the menstrualcycle to be male if the value of HORAC/(E/T) from step (c) is less thanabout 150, or to be female if the value from step (c) is greater thanabout 200.

The present invention also provides for a method of determining genderspecific compatibility of an ovum released in a menstrual cycle forpre-conception baby gender planning comprising the steps of: (a)performing hydroxyl radical antioxidant capacity (HORAC) assay on a bodyfluid from a non-pregnant female; (b) measuring levels of testosterone(T), estrogens (E), progesterone (P) and gonadotropins (G) in the bodyfluid, wherein step (b) can be performed before or after step (a); (c)calculating the value of HORAC/[E−(T+P+G)], wherein [E−(T+P+G)] is thedifference between the level of estrogens (E) and the total levels oftestosterone (T), progesterone (P) and gonadotropins (G), and whereinthe value of HORAC/[E−(T+P+G)] is the ratio of the HORAC value to the[E−(T+P+G)] value; (d) comparing the HORAC/[E−(T+P+G)] value from step(c) with at least one standard; and (e) determining the gender specificcompatibility of the ovum released in the menstrual cycle to be male ifthe HORAC/[E−(T+P+G)] value from step (c) falls within a range of thestandard with a male gender specific compatibility, or to be female ifthe HORAC/[E−(T+P+G)] value from step (c) falls within a range of thestandard with a female gender specific compatibility.

The present invention further provides for a method of determininggender specific compatibility of an ovum released in a menstrual cyclefor pre-conception baby gender planning comprising the steps of: (a)performing hydroxyl radical antioxidant capacity (HORAC) assay on a bodyfluid from a non-pregnant female; (b) measuring levels of testosterone(T), estrogens (E), progesterone (P) and gonadotropins (G) in the bodyfluid, wherein step (b) can be performed before or after step (a); (c)calculating the value of HORAC/[(E+T)/(P+G)] wherein [(E+T)/(P+G)] isthe ratio of the total levels of estrogens (E) and testosterone (T) tothe total levels of progesterone (P) and gonadotropins (G), and whereinthe value of HORAC/[(E+T)(P+G)] is the ratio of the HORAC value to the[(E+T)/(P+G)] value; (d) comparing the HORAC/[(E+T)/(P+G)] value fromstep (c) with at least one standard; and (e) determining the genderspecific compatibility of the ovum released in the menstrual cycle to bemale if the HORAC/[(E+T)/(P+G)] value from step (c) falls within a rangeof the standard with a male gender specific compatibility, or to befemale if the HORAC/[(E+T)/(P+G)] value from step (c) falls within arange of the standard with a female gender specific compatibility.

Further provided in the present invention is a method of determininggender specific compatibility of an ovum released in a menstrual cyclefor pre-conception baby gender planning comprising the steps of: (a)contacting a body fluid from a non-pregnant female with free radicals;(b) measuring free radical scavenging activity of the body fluid; (c)comparing the free radical scavenging activity of the body fluid with atleast one standard; and (d) determining gender specific compatibility ofthe ovum released in a menstrual cycle.

Further provided in the present invention is a method of determininggender specific compatibility of an ovum released in a menstrual cyclefor pre-conception baby gender planning comprising the steps of: (a)contacting a body fluid from a non-pregnant female with a solid surfacehaving antibodies specific to estrogens, antibodies specific totestosterone, antibodies specific to progesterone and antibodiesspecific to hCG-beta; (b) contacting the solid surface from step (a)with at least one redox indicator; (c) measuring redox activity of thesolid surface; (d) comparing the redox activity of the solid surfacewith at least one standard; and (e) determining the gender specificcompatibility of the ovum released in the menstrual cycle.

The present invention provides for a method of conceiving a baby of adesired gender in a female comprising the step of applying to the femalea pharmaceutical formulation selected from the group consisting of: (i)a pharmaceutical formulation comprising about 1 ng/ml to about 35 ng/mlestradiol, about 40 ng/ml to about 600 ng/ml estriol, about 0.1 ng/ml toabout 35 ng/ml estrone, about 1 ng/ml to about 25 ng/ml testosterone,about 1 ng/ml to about 500 ng/ml progesterone when the desired gender ofthe baby is male, wherein the [E−(T+P)] value ranges from about 300 toabout 1,500, and (ii) a pharmaceutical formulation comprising about 1ng/ml to about 20 ng/ml estradiol, about 80 ng/ml to about 500 ng/mlestriol, about 1 ng/ml to about 20 ng/ml estrone, about 5 ng/ml to about80 ng/ml testosterone, about 100 ng/ml to about 1000 ng/ml progesteronewhen the desired gender of the baby is female, wherein the [E−(T+P)]value ranges from about −10 to about −800. The pharmaceuticalformulation may be in the form of a gel, a solution, a cream, a lotion,an ointment, a foam or a paste.

DETAILED DESCRIPTION

The present invention provides methods for determining the gender of anunborn child as well as preconception fetal gender planning by assayingvarious sex hormones, evaluating the overall redox activity, and/orevaluating radical scavenging capacity of the maternal urine or anon-pregnant female's urine. The methods may be conducted in other bodyfluids such as tears and cerebrospinal fluid. The method can be used todetermine fetal gender at any time point during the entire pregnancy,i.e., from about four days before menstrual due date to about 40 weeksof pregnancy. In one embodiment, the body fluid is obtained from apregnant female between the 4th week and 15th week of pregnancy. Inorder to improve accuracy of the results, the body fluid may beprocessed before assaying. Processing may involve aging the body fluid,or purification of various fractions. For example, processing cancomprise aging the urine by storing it at ambient temperature (e.g.,approximately, 20-30° C.) for a defined period of time, e.g., 1-4 weeks,treating the urine by physical, chemical or biochemical means toaccelerate the aging process, and/or removing various urinary componentsby physical, chemical or biochemical means. Repeated analysis of urinesamples obtained from the same test subject at different time points inthe pregnancy appears to improve the accuracy of the analytical results.For example, if three urine samples are analyzed, we have found that theaccuracy of the results approaches 100%.

The methods of the present invention also provide for a means forpre-conception baby gender planning by assaying the sex hormones,evaluating the overall redox activity, and/or evaluating radicalscavenging capacity of a non-pregnant female's urine or other bodyfluid. The body fluid may be assayed at anytime during a menstrualcycle, but preferably is assayed around the time of ovulation or middleof the menstrual cycle. Without being limited to any specificphysiological mechanism, it is believed that ovulation generates atleast two types of uterine environments during alternate menstrualcycles, presumably due to the release of two different types of ova. Onetype of ovum is associated with uterine environments to be compatiblewith fertilization by a Y chromosome-bearing sperm to produce a malefetus, whereas the other type of ovum is associated with uterineenvironments to be compatible with fertilization by an Xchromosome-bearing sperm to produce a female fetus. The fertilizationrate for a sperm and an ovum in incompatible environments is very low.We have found that sex hormone levels, the overall redox activity,and/or radical scavenging capacity of a urine sample correlate with thegender specific compatibility of the ovum being released during aparticular menstrual cycle. Therefore, assaying sex hormone levels, theoverall redox activity, and/or radical scavenging capacity of anon-pregnant female's urine will help a couple conceive a baby of adesired gender.

The methods of the present invention may encompass assaying any suitablebody fluid, including urine, tears, saliva, sweat, blood, plasma, serum,cerebrospinal fluid and amniotic fluid. Pigman et al. The reducing powerof human saliva and its component secretions. J. Dent Res. 37(4):688-696 (1958). Oyawoye et al. Antioxidants and reactive oxygen speciesin follicular fluid of women undergoing IVF. Human Reproduction. 18(11): 2270-2274 (2003).

The term “overall redox activity” or “redox activity” of a body fluidrefers to the net redox activity (including antioxidant activity and/orradical scavenging capacity) of a body fluid as a whole, althoughvarious components of the body fluid may exhibit different redoxactivities individually. The overall redox activity of a body fluid suchas urine depends not only on the composition of the urine, but also, onhow the sample is processed before assaying, as well as on the assayconditions. For example, the body fluid may be “aged” for at least about1 week at room temperature ranging from about 20° C. to about 30° C.before assaying. The body fluid may also be aged for longer periods oftime such as about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 and 12 months. Thebody fluid may be aged at room temperature for a period of time rangingfrom about 1 week to about 52 weeks prior to assaying. Aging of theurine sample can range from as long as 1 year to about 5 years, butlonger periods may also be appropriate. Other suitable chemical,biochemical or physical treatments may also be used to process the urinebefore assaying.

The term “fetal gender related characteristics” refers to (i) theevaluation of the type of uterine environment and compatibility of ovumreleased in a particular menstrual cycle before pregnancy, and (ii) thedetermination of sex of the fetus after pregnancy. Accordingly, “malegender related characteristics” refers to (i) a uterine environmentand/or ovum released in a particular menstrual cycle compatible tofertilize a Y chromosome-bearing sperm before pregnancy, and (ii) a maleunborn baby. Similarly, “female gender related characteristics” refersto (i) a uterine environment and/or ovum released in a particularmenstrual cycle compatible to fertilize an X chromosome-bearing spermbefore pregnancy, and (ii) a female unborn baby.

The term “gender specific compatibility” refers to the compatibility ofan ovum (or uterus) for fertilization by either a Y chromosome-bearingsperm to produce a male fetus, or by an X chromosome-bearing sperm toproduce a female fetus. When an ovum (or uterus) is compatible withfertilization by a Y chromosome-bearing sperm to produce a male fetus,the gender specific compatibility of the ovum (or uterus) is male; whenan ovum (or uterus) is compatible with fertilization by an Xchromosome-bearing sperm to produce a female fetus, the gender specificcompatibility of the ovum (or uterus) is female.

Estrogens include, but are not limited to, estradiol, estriol, estrone,their metabolites and derivatives. Sex hormones include, but are notlimited to, estrogens, androgens, progestogens, hCG, follicularstimulating hormone, leutenising hormones, glycoproteins, as well astheir metabolites, breakdown products and derivatives. Androgens mayinclude testosterone, dihydrotestosterone, dehydroepiandrosterone andandrostenedione. Progestogens include all steroids with a pregnaneskeleton, such as progesterone.

The present invention provides a method of determining the gender of anunborn child comprising the steps of: (a) measuring levels oftestosterone (T), estrogens (E), progesterone (P) and gonadotropins (G)in a body fluid from a pregnant female; (b) calculating [E−(T+P+G)] asthe difference between the level of estrogens (E) and the total levelsof testosterone (T), progesterone (P) and gonadotropins (G); (c)comparing the [E−(T+P+G)] value from step (b) with at least onestandard; and (d) determining the gender of the unborn child. The sexhormones, such as testosterone, estrogens, progesterone andgonadotropins, may be attached to a solid surface before step (a).

In one embodiment, there is provided a method of determining the genderof an unborn child comprising the steps of: (a) measuring levels oftestosterone (T), estrogens (E), progesterone (P) and gonadotropins (G)in a body fluid from a pregnant female; (b) calculating [E−(T+P+G)] asthe difference between the level of estrogens (E) and the total levelsof testosterone (T), progesterone (P) and gonadotropins (G); and (c)determining the gender of the unborn child to be male if the [E−(T+P+G)]value from step (b) ranges from about 300 to about 1,500, or to befemale if the [E−(T+P+G)] value from step (b) ranges from about −10 toabout −800.

Because the measured levels of the sex hormones may depend on theparticular reagents and method that is used to measure the sex hormonelevels, the mathematical relationship of the various sex hormones in thebody fluid from a test subject may be compared with that of a standard,i.e., a body fluid from a pregnant female where the sex of the fetus isknown, to determine the gender of the fetus carried by the test subject.Alternatively, the mathematical relationship of the various sex hormonesin the body fluid from a test subject is compared with the mathematicalrelationship of the various sex hormones of the body fluid samples frompregnant females where the sexes of the fetuses are known, to determinethe gender of the fetus carried by the test subject. Similarly, forpre-conception methods, the mathematical relationship of the various sexhormones of the body fluid from a test subject is compared with that ofa standard, i.e., body fluid from a non-pregnant female where the genderspecific compatibility of the ovum the female generated is known, todetermine the gender specific compatibility of the ovum produced by thetest subject. Alternatively, the mathematical relationship of thevarious sex hormones of the body fluid from a test subject is comparedwith the mathematical relationship of the various sex hormones of thebody fluid samples from non-pregnant females where the gender specificcompatibilities of the ova the females generated are known, to determinethe gender specific compatibility of the ovum produced by the testsubject. The mathematical relationship of the various sex hormones ofthe standard may be determined using any method encompassed by thisinvention. One standard may be used in the present invention.Alternatively, more than one standard may be used. The standard may beat least one body fluid sample from one female, or may be at least onebody fluid sample from more than one female where the sex of the fetusor the gender specific compatibility of the ovum is known. Thus, thevalue of the standard can be a range corresponding either to a femalefetus (or female gender specific compatibility of the ovum) or a malefetus (or male gender specific compatibility of the ovum).Alternatively, more than one body fluid sample from one female, or atleast one body fluid sample from more than one female, can be pooled,and the standard value determined.

The level of a sex hormone can be measured by any suitable method knownto those skilled in the art. For example, the level of a sex hormone canbe measured by specific antibodies or antigen-binding fragments. Themeasurement may thus comprise a step of contacting the body fluid with,for example, testosterone-specific, estrogens-specific,progesterone-specific and/or gonadotropins-specific antibodies.

A detectable label may be conjugated to the antibody or antigen-bindingfragment. A detectable label may be conjugated to a primary antibodyand/or a secondary antibody. Exemplary detectable labels include aradioisotope, a fluorophore, a luminescent molecule, an enzyme, a biotinmoiety, an epitope tag, a dye molecule, and a molecule capable ofactivating a chemiluminescent substrate. Exemplary enzyme labels includephosphatases (such as, alkaline phosphatase (ALP)) and peroxidases(e.g., horseradish peroxidase (HRP)). These and others will be apparentto one of ordinary skill in the art. WO2009108307.

The level of the sex hormone associated with the antibodies may bedetermined by any suitable assay, such as enzyme-linked immunosorbentassay (ELISA), immunoassay (including enzyme immunoassay),radioimmunoassay, immunoradiometric assay, immunochemiluminescent assay,Western blot, immunoprecipitation, and surface plasmon resonance (SPR)immunoassay. See, for example, WO 2009033212. In one embodiment,testosterone enzyme immunoassay test kit and estradiol (E2) enzymeimmunoassay test kit from BioCheck, Inc. may be used to determinetestosterone and estradiol levels. Levels of different sex hormones canbe measured by the same method or by different methods. WO 2008067008discloses using SPR technology to simultaneously and quantitativelymeasure the concentrations of different sex hormones in a sample.

When level of the sex hormone is measured by specific antibodies, theantibodies may be provided in a solution or may be attached to a solidphase. The solid phase may be a particle, bead, cover slip, slide, tube,microtiter well, sheet, chip, reaction tray, strip, membrane, film,fiber, plate, bottle, box, or any other shape/material suitable for thepresent invention. The solid surface may be of any material or shapecapable of supporting chemical reactions.

When the solid phase is a particle, the particle may be a microparticleor nanoparticle. The diameter of the particle may be from about 0.1 nmto about 100 mm, from about 10 nm to about 50 mm, from about 100 nm toabout 30 mm, from about 500 nm to about 20 mm, from about 0.1 nm toabout 100 nm, or from about 700 nm to about 10 mm The solid phase canconsist of natural, semi-synthetic or synthetic materials. The solidphase can be made of biodegradable and/or non-biodegradable materials.Non-biodegradable materials include, but are not limited to, silica,latex, glass, quartz, metal (e.g., nickel, gold, colloidal gold), mica,plastic, derivatized plastic, ceramic, carbon, bentonite, alumina,borosilicate, zeolites, natural or synthetic composites of redoxsensitive materials like iron, copper, chromium, manganese, tungsten,molybdenum, vanadium, arsenic, germanium, or combinations thereof. Thecarrier matrix can be made of polyester, polycarbonate, polysulfone,polyvinyl chloride, polyethylene, polypropylene, poly(N-vinylpyrrolidone), poly(methyl methacrylate), poly(vinyl alcohol),poly(acrylic acid), polyacrylamide, poly(ethylene-co-vinyl acetate),poly(methacrylic acid), and combinations thereof. Biodegradablematerials include, but are not limited to, starch, cross-linked starch,poly(ethylene glycol), polyvinylpyrrolidine, polylactides (PLA),polyglycolides (PGA), poly(lactide-co-glycolides) (PLGA),polyanhydrides, polyorthoesters, poly(DTH iminocarbonate),poly(bisphenol A iminocarbonate), polycyanoacrylate, polyphosphazene,mixtures thereof and combinations thereof. European Patent ApplicationNos. 0184899 and 0186947. The solid phase may contain polystyrene,polysuflone, polyurethane, and polytetrafluoroethylene (PTFE),polyacrylate and their derivatives, copolymers (e.g. Eupergit andDynospheres), protein (such as gelatin), polysaccharides (such asdextran, agarose, cellulose, nitrocellulose), acrylic, or mixturesthereof.

The solid phase may also comprise additive agent that providesadditional properties to the particle. Such additives include magneticmaterial, or a fluorescent compound.

In certain embodiments, the antibodies are attached to a particle havinga fluorescent compound positioned within or on the surface of theparticle. The antibodies may be attached directly to the fluorescentcompound. Antibodies with different specificity may be associated withfluorescent compounds having emission spectra distinguishable from eachother, allowing for binding of different sex hormones to the particleand for different sex hormones to be distinguished. Labeling multipleantibodies with different fluorescent compounds allows measurement ofmultiple hormones simultaneously. For example, the fluorescent compoundsattached to testosterone-specific antibodies, estrogens-specificantibodies, progesterone-specific antibodies and gonadotropins-specificantibodies may all be different.

In certain embodiments, there are a fluorescent compound positionedwithin a particle attached to specific antibodies, where thespecificities of the antibodies attached to one particle are the same.For example, a particle only contains antibodies specific totestosterone, estrogens, progesterone or gonadotropins. The fluorescentcompound of the particle attached to testosterone-specific antibodies,the fluorescent compound of the particle attached to estrogens-specificantibodies, the fluorescent compound of the particle attached toprogesterone-specific antibodies, and the fluorescent compound of theparticle attached to gonadotropins-specific antibodies are alldifferent.

The fluorescent compounds that may be used in this invention can coverthe entire UV-V is to near-IR absorption and emission spectrum. Anyknown fluorescent compound may be used, such as fluorescent organiccompound, dyes, pigments, or combinations thereof. A wide variety ofsuitable fluorescent dyes are known, see, for example, Molecular ProbesHandbook of Fluorescent Probes and Research Chemicals, 6th ed., R. P.Haugland, ed. (1996). A typical fluorophore is, for example, afluorescent aromatic or heteroaromatic compound such as is a pyrene, ananthracene, a naphthalene, an acridine, a stilbene, an indole orbenzindole, an oxazole or benzoxazole, a thiazole or benzothiazole, a4-amino-7-nitrobenz-2-oxa-1,3-diazole (NBD), a cyanine, a carbocyanine,a carbostyryl, a porphyrin, a salicylate, an anthranilate, an azulene, aperylene, a pyridine, a quinoline, a coumarin (includinghydroxycoumarins and aminocoumarins and fluorinated derivativesthereof), and like compounds, see for example U.S. Pat. Nos. 5,830,912,4,774,339, 5,187,288, 5,248,782, 5,274,113, 5,433,896, 4,810,636 and4,812,409.

Various kinds of fluorophores are known in the art, including, but notlimited to, fluorescein isothiocyanate (FITC), tetramethylrhodamineisothiocyanate (TRITC), phycoerythrin, Cy7, fluorescein (FAM), Cy3,Cy3.5 (also known as Cy3++), Texas Red, LightCycler-Red 640, LightCyclerRed 705, tetramethylrhodamine (TMR), rhodamine, rhodamine derivative(ROX), hexachlorofluorescein (HEX), Cy5, Cy5.5 (also known as Cy5++),Cy2, rhodamine 6G (R6G), the rhodamine derivative JA133, AlexaFluorescent Dyes (such as Alexa Fluor 488, Alexa Fluor 546, Alexa Fluor633, Alexa Fluor 555, and Alexa Fluor 647),4′,6-diamidino-2-phenylindole (DAPI), Propidium iodide, AMCA, SpectrumGreen, Spectrum Orange, Spectrum Aqua, Lissamine, and fluorescenttransition metal complexes, such as europium. Fluorophores that can beused also include fluorescent proteins, such as GFP (green fluorescentprotein), enhanced GFP (EGFP), blue fluorescent protein and derivatives(BFP, EBFP, EBFP2, Azurite, mKalamal), cyan fluorescent protein andderivatives (CFP, ECFP, Cerulean, CyPet) and yellow fluorescent proteinand derivatives (YFP, Citrine, Venus, YPet). WO2008142571, WO2009056282,WO9922026.

The particles of the present invention may exhibit intrinsicfluorescence. U.S. Pat. No. 6,207,392. Bruchez et al., SemiconductorNanocrystals as Fluorescent Biological Labels. Science, 281, 2013(1998). Particles that are able to bind one type of sex hormone may havesame or similar diameter, which can be different from the diameter ofthe particles binding to a different type of sex hormone. Particleshaving different diameters may have different emission spectradistinguishable from each other. For example, nanoparticles of differentdiameters may exhibit different optical color. In one embodiment,various antibodies for different sex hormones are attached to a solidphase. After the body fluid, e.g., urine, is added to the solid phase,sex hormones will bind to their specific antibodies attached to thesolid phase. When the antibody-linked particles are added to the surfaceof the solid phase, the intensity of the emission of the particles witha specific emission spectrum will correspond to the level of a sexhormone. Accordingly, the fetal gender related characteristics such asthe gender of an unborn baby can be determined by the overall emissionspectrum or the overall optical color of the solid phase. Alternatively,the antibodies attached to the particle may be conjugated with at leastone redox indicator. The fetal gender related characteristics such asthe gender of an unborn baby can then be determined by the overall redoxactivity of the solid phase.

In another embodiment, the body fluid, e.g., urine, is added to theparticles. Different sex hormones in the body fluid will then attach orbe adsorbed to particles exhibiting different spectral or opticalproperties (for example, exhibiting different colors). The particleswith the attached (or adsorbed) sex hormones are added to a solid phaseto which various antibodies for different sex hormones are attached.Then, the overall color exhibited by such a solid surface can be used toevaluate fetal gender related characteristics. Alternatively, theantibodies attached to the particle may be conjugated with at least oneredox indicator, and the overall redox activity of the solid phase canbe determined by reacting such a solid surface with a redox indicator,thereby revealing fetal gender related characteristics.

The level of the sex hormone may be measured by any other suitabletechnique known to a skilled artisan. The techniques include, but arenot limited to, mass spectrometry (MS), high performance liquidchromatography (HPLC), gas chromatography (GC), GC-MS, affinitychromatography, ion exchange chromatography, size exclusionchromatography, and reversed-phase chromatography, nuclear magneticresonance (NMR) spectroscopy, infrared spectroscopy (IR), thin layerchromatography (TLC), and multiplex array technology.

The levels of different sex hormones, such as testosterone, estrogens,progesterone and gonadotropins, may be measured in separate assays, ormay be measured simultaneously or sequentially in one assay.

The present invention provides a method of determining the gender of anunborn child comprising the steps of: (a) measuring levels oftestosterone (T), estrogens (E), progesterone (P) and gonadotropins (G)in a body fluid from a pregnant female; (b) calculating (E+T)/(P+G) asthe ratio of the total levels of estrogens (E) and testosterone (T) tothe total levels of progesterone (P) and gonadotropins (G); and, (c)comparing the ratio (E+T)/(P+G) from step (b) with at least one standardto determine the gender of the unborn child.

In one embodiment, there is provided a method of determining the genderof an unborn child comprising the steps of: (a) measuring levels oftestosterone (T), estrogens (E), progesterone (P) and gonadotropins (G)in a body fluid from a pregnant female; (b) calculating (E+T)/(P+G) asthe ratio of the total levels of estrogens (E) and testosterone (T) tothe total levels of progesterone (P) and gonadotropins (G); and (c)determining the gender of the unborn child to be male if the (E+T)/(P+G)ratio from step (b) is greater than about 1.4, or to be female if the(E+T)/(P+G) ratio from step (b) is less than about 1.2.

The present invention provides a method for determining the gender of anunborn child comprising the steps of: (a) contacting a body fluid from apregnant female with at least one redox indicator; (b) measuring redoxactivity of the body fluid; and, (c) comparing the redox activity of thebody fluid with at least one standard to determine the gender of theunborn child.

Because the ability of the body fluid to reduce or oxidize a reagentwill depend on the particular reagent (reducing agent or oxidizingagent) that is used to measure reduction or oxidation, the redoxactivity of the body fluid from a test subject is compared with theredox activity of a standard, i.e., the body fluid from a pregnantfemale where the sex of the fetus is known, to determine the gender ofthe fetus carried by the test subject. Alternatively, the redox activityof the body fluid from a test subject is compared with the redoxactivity of the body fluid samples from pregnant females where the sexesof the fetuses are known, to determine the gender of the fetus carriedby the test subject. Similarly, for pre-conception methods, the redoxactivity of the body fluid from a test subject is compared with theredox activity of a standard, i.e., body fluid from a non-pregnantfemale where the gender specific compatibility of the ovum the femalegenerated is known, to determine the gender specific compatibility ofthe ovum produced by the test subject. Alternatively, the redox activityof the body fluid from a test subject is compared with the redoxactivity of the body fluid samples from non-pregnant females where thegender specific compatibilities of the ova the females generated areknown, to determine the gender specific compatibility of the ovumproduced by the test subject. The redox activity of the standard may bedetermined using any method encompassed by this invention. One standardmay be used in the present invention. Alternatively, more than onestandard may be used.

The overall redox activity or the mathematical relationship of thevarious sex hormones of the body fluid such as urine may depend not onlyon the composition of the urine, but also, on how the sample isprocessed before assaying, as well as on the assay conditions. Forexample, if the urine sample is not aged or is assayed under mildconditions (see description below), the overall redox activity of theurine correlates with its in vivo overall redox activity. Namely, theurine from a female carrying a female fetus will have a comparativelyhigher overall reducing activity (or antioxidant activity, or radicalscavenging activity) than urine from a female carrying a male fetus.

A similar observation has been made for pre-conception assays. Namely,if the urine sample is not aged or is assayed under mild conditions, theurine sample of a non-pregnant female carrying an ovum compatible withfertilization by an X chromosome-bearing sperm will have a comparativelyhigher reducing activity (or antioxidant activity, or radical scavengingactivity) than urine from a non-pregnant female carrying an ovumcompatible with fertilization by a Y chromosome-bearing sperm.

In contrast, if the urine sample is allowed to age and/or is assayedunder harsh conditions (see description below), the urine would exhibitan overall redox activity different from its activity in vivo. Namely,the urine from a female carrying a male fetus will have a comparativelyhigher overall reducing activity than urine from a female carrying afemale fetus.

Likewise, with pre-conception testing, if the urine sample is assayedunder harsh conditions, the urine sample of a non-pregnant femalecarrying an ovum compatible with fertilization by a Y chromosome-bearingsperm will have a comparatively higher reducing activity than urine froma non-pregnant female carrying an ovum compatible with fertilization byan X chromosome-bearing sperm. See Table 1 for the redox activities ofthe urine samples under different processing and assay conditions.

TABLE 1 Testoster- one:Estrogens Urine not aged or assayed Urine aged orassayed Ratio under mild conditions under harsh conditionsPost-conception Male Fetus Greater than Comparatively higherComparatively higher about 1 overall oxidizing overall reducing (oractivity antioxidant) activity Female Fetus Less than about 1Comparatively higher Comparatively higher overall reducing (or overalloxidizing antioxidant) activity activity Pre-conception Ovum compatibleGreater than Comparatively higher Comparatively higher with Ychromosome- about 1 overall oxidizing overall reducing (or bearing spermactivity antioxidant) activity Ovum compatible Less than about 1Comparatively higher Comparatively higher with X chromosome- overallreducing (or overall oxidizing bearing sperm antioxidant) activityactivity

The term “mild condition” refers to processing of the body fluid priorto assaying and assaying of the body fluid by treatment at temperatureslower than about 21° C. or treatment with mild chemicals, such as talc,alumina, extraction using organic solvents, solid phase extraction (SPE)chromatography or phosphotungstic acid (PTA). The term “harsh condition”refers to processing of the body fluid prior to assaying or assaying ofthe body fluid by treatment at temperatures greater than about 21° C. ortreatment by harsh chemicals, such as strong acids, strong alkalis,trichloroacetic acid and strong oxidizing agents. Strong oxidizingagents include potassium ferricyanide in the ferric reducing/antioxidantpower (FRAP) assay. The particular condition of the processing of thebody fluid prior to assaying or assaying of the body fluid can bedetermined by one of ordinary skill in the art without undueexperimentation.

There are a number of reducing or oxidizing compounds present in theurine that may interfere with the assays of the present invention.Therefore, the urine may be processed to remove these compounds beforeassaying the redox activity. Interfering reducing and oxidizing agentsinclude urea, thiourea, creatine, uric acid, ascorbic acid, glucose,glucuronic acid, bilirubin, creatinine, porphyrins and related pigments,nitrites, sulfites, bisulfites, hyposulfites, pyrosulfites, sulfatesoxyacids (such as sulfurous acid, bisulfurous acid, hyposulfurous acid,pyrosulfurous acid), urobilinogen, hemoglobin or other interferingproteins, and leucocytes. Interfering agents can be removed by treatmentwith enzymes including ascorbic acid oxidase, glucose oxidase, ureaseand uricase. Glucose may be removed by an anion exchange resin such asAmberlite-4B or alumina. Interfering agents may also includeprogesterone, its derivatives and/or metabolites, which may be removedby adsorbents such as mica and highly oriented pyrolytic graphite, or byantibodies against progesterone, its derivatives and/or metabolites.Ascorbic acid can be removed by resin extraction, diazotized4-nitroaniline-2,5-dimethoxyaniline, ascorbic acid oxidase, leadacetate, iodate, and/or their combinations. Michaelson. Scand J Clin LabInves 20: 97 (1967). Hughes. Analyst, 89:618 (1964).

Chemical, biochemical or physical treatments may also be used to removethe interfering reducing and/or oxidizing compounds before assaying. Thetreatments may include extraction, purification, adsorption, andtreatments by mineral acids, enzymes or microwaving. The body fluid mayalso be processed by treatment with adsorbants and/or precipitants.Adsorbants may be selected from the group consisting of talc,silica-based particles such as silica gel, alumina, florisil, charcoal,kaolin, concanavaline A and its conjugates, calcium phosphate, calciumhydroxide, calcium chloride, Cetyltrimethyl ammonium bromide, lectinspecific to the carbohydrate portion of urinary gonadotropins orglycoproteins, protein or glycoprotein hydrolyzing enzymes, glassfiberfilter, ion exchange resins, affinity ligands, extraction using organicsolvents, solid phase extractants, size exclusion sieves, and reversephase chromatographic materials. Precipitants may be salts of heavymetals, which are selected from the group consisting of barium, lead,molybdenum and tungsten. Precipitants may comprise barium chloride,barium hydroxide mixed with zinc chloride, mercuric chloride, leadacetate and their mixtures. Precipitants may also comprise ammoniumsulphate, dextran, acetonitrile, chloroform, sodium hydroxide,trichloroacetic acid, potassium iodate and their mixtures. One or morethan one adsorbant or precipitant may be used; in addition, adsorbantand precipitant may be used in combination. Adsorbants or precipitantsmay be used to adsorb or retain the interfering reducing or oxidizingagents. Alternatively, adsorbants or precipitants may be used to adsorbor retain desired urinary fraction, which may later be eluted withappropriate solvents for assaying.

When the urine sample is allowed to age, most of the interferingreducing or oxidizing components appear to be eliminated. In oneembodiment, the urine sample is first processed by physical, chemical orbiochemical means to accelerate the aging process. The urine sample isthen treated by other chemical, physical or biochemical means to obtaina purified or partially purified fraction, followed by assaying theoverall redox activity of the urine sample under harsh conditions.

Prior to assaying, insoluble components may be removed from the bodyfluid, for example, by filtration or centrifugation. A non-interferingpH indicator may also be added prior to assaying. The pH of the bodyfluid may be adjusted.

The primary hormones directly linked to fetal gender are sex hormones.The ratio of testosterone to estrogens is greater than about 1.0 in theurine from a mother carrying a male fetus, whereas the ratio oftestosterone to estrogens is less than about 1.0 in the urine from amother carrying a female fetus. The same may be true for midcycle urinesamples from non-pregnant women. Sex hormones, including testosteroneand estrogen, are excreted in urine as their glucuronides associatedwith large glycoproteins such as human chorionic gonadotropin (hCG), sexhormone-binding globulin, as well as other glycoproteins. In freshlyvoided urine, sex hormones maintain their association with these largemolecules. Without being limited to any specific physiologicalmechanism, it is believed that the overall redox activity of a urinesample assayed under mild conditions or without aging is the net redoxactivity of various urinary complexes which comprise sex hormones andlarge molecules such as hCG, sex hormone-binding globulin, as well asother glycoproteins.

When urine is assayed under harsh conditions, the hormones which cancontribute to the reducing activity of a urine sample are progesterone,testosterone and human chorionic gonadotropin (hCG) and or itsdegradation byproducts, whereas estrogens can contribute to theoxidizing activity of the urine. hCG is a glycoprotein hormonespecifically produced in pregnancy that is secreted by the embryo soonafter conception and later by the placenta. hCG's major role is tomaintain progesterone production that is critical for maintaining apregnancy in humans. Many currently available early pregnancy tests arebased on the detection or measurement of hCG in the blood or urine. Theurine or blood level of hCG itself does not correlate with the gender ofthe fetus. hCG exhibits reducing activity. Nepomnaschy et al. HumanReproduction. 2008. 23(2): 271. Under harsh assay conditions, if a urinesample is from a female carrying a male fetus, there is no otherstronger reducing agent (or antioxidant) in the urine sample that isable to protect hCG from being oxidized by urinary oxidizing agents.Bowman D. E. J. Biol. Chem. 1940, p 293-302. Gurin et al. J. Biol. Chem.1940. 128: 525. As a result, hCG is oxidized and subsequently degradedto its constituents including galactoses and hexoamines, both of whichhave much stronger reducing activities than hCG. As a result, galactosesand hexoamines contribute to a comparatively higher overall reducingactivity of the urine from a female carrying a male fetus in comparisonwith urine from a female carrying a female fetus. In contrast, in afreshly voided urine sample without aging or under mild assayconditions, if a urine sample is from a female carrying a female fetus,a relatively higher concentration of estrogen in the urine sample isable to protect hCG from oxidizing agents by interacting with theoxidizing agents. As a result, hCG is not oxidized or degraded to thesame extent. Rather, hCG is degraded at a much slower rate in comparisonto hCG in the urine sample from a female carrying a male fetus.Therefore, after aging or under harsh conditions, the urine from afemale carrying a male fetus would exhibit a higher overall reducingactivity than the urine from a female carrying a female fetus.

In samples from non-pregnant women, hCG may play a less significantrole, whereas other glycoproteins like FSH and LH may be more important.

Sex hormones are excreted in urine as their glucuronide conjugates. Asthe urine sample is aged, glucuronide conjugates are hydrolyzed and freesex hormones released. To facilitate release of the free sex hormonesfrom their glucuronide conjugates, a urine sample may be treated withthe enzyme β-glucuronidases. β-glucuronidases purified from varioussources can be used, including bovine liver, snail Helix pomatia and E.coli. In one embodiment, the urine sample is treated withβ-glucuronidases from E. coli for 24 hours. In another embodiment, theurine sample is treated with E. coli β-glucuronidases for 2 hours.

The urine sample may also be treated by a mineral acid to facilitaterelease of the free sex hormones from their glucuronide conjugates. Themineral acid may be hydrochloric acid or sulfuric acid. The yield ofhydrolysis is determined by the property and concentration of the acid.In one embodiment, a complete hydrolysis of the sex hormone glucuronideconjugates is achieved by treating a urine sample with 3 M sulfuric acidat 37° C. for 24 hours.

The redox potential of the body fluid may also be assayed. Redoxpotential may be determined by measuring the potential differencebetween an inert indicator electrode in contact with the body fluid anda stable reference electrode connected to the body fluid by a saltbridge. The inert indicator electrode acts as a platform for electrontransfer to or from the reference half cell. The indicator electrode maycomprise platinum. The indicator electrode may comprise gold andgraphite. The reference electrode may be stable hydrogen electrode(SHE), Ag/AgCl reference electrode or saturated calomel (SCE) referenceelectrode.

Any suitable assay method, or a combination of two or more such assaymethods, capable of measuring the overall redox activity of the bodyfluid may be used with the methods of the present invention. The overallredox activity of the body fluid may be assayed by reacting the bodyfluid with at least one redox indicator. As used herein, the term “redoxindicator” refers to a molecule that undergoes a significant measurablechange upon being reduced or oxidized by exchanging electron, proton orhydrogen atom with any biochemical component of the body fluid underexamination.

A redox indicator may be a reducing agent, oxidizing agent or radical.The measurable change may be changes in color, fluorescence,chemiluminescence, electromagnetic radiation or any other suitablechanges that may be assayed. The amount of the redox indicator reducedor oxidized may be directly correlated with the overall redox activityof the body fluid, which in turn is correlated with fetal gender, or thetype of the ovum being produced. The redox indicator may be achromogenic chemical capable of changing color upon being reduced oroxidized. The amount of the chromogenic chemical being reduced oroxidized can be measured by the difference in absorbance of the bodyfluid at a specific wavelength before and after the reduction/oxidationreaction.

Below are presented various types of redox assays that may be used todetermine the overall redox activity of the body fluid such as urine.The overall redox activity of the body fluid may be determined bycomparison with at least one standard where the sex of the fetus isknown, or where the gender specific compatibility of the ovum is known.The comparative levels of the redox activity of the urine samples areshown in Table 1. The particular condition in each assay may bedetermined by a person of ordinary skill in the art without undueexperimentation.

The overall reducing activity of a body fluid may be assayed using aheteropoly acid or its corresponding salts. Heteropoly acids are a classof acids each comprising a particular combination of metal, hydrogen,oxygen and other non-metal atoms. Specifically, a heteropoly acidcontains a metal termed addenda atom, such as tungsten, molybdenum orvanadium; oxygen; a element termed hetero atom generally from thep-block of the periodic table, such as silicon, phosphorus or arsenic;and acidic hydrogen atoms. Two of the better known heteropoly acids arephosphotungstic acid (PTA) with the formula H3PW₁₂O₄₀ andphosphomolybdic acid with the formula H₃PMo₁₂O₄₀.

The phosphotungstic and phosphomolybdic acids are very sensitive toreduction, and yield highly colored compounds even upon moderatereduction. Wu. Contributions to the chemistry of phosphomolybdic acid,phosphotungstic acid and allied substances. J. Biol. Chem. 1920. XLIII,1: 189-220. Under acidic condition, PTA associates with proteins andforms precipitates in solution. Phosphotungstate: a “universal”(nonspecific) precipitant for polar polymers in acid solution. Journalof Histochemistry and Cytochemistry. J. E. Scott. 1971. 19, 11, 689.Besides proteins, PTA also has an affinity for carbohydrates at low pH.Pease D C. J. Ultrastructure Res. 1966. 15: 555. As pH of its solutionincreases, a heteropoly acid generally decomposes to its simplerconstituent acids, but can be regenerated by re-acidification.

The assay using PTA may be conducted under mild conditions. With a PTAassay, the urine from a female carrying a female fetus will have acomparatively higher overall reducing activity as compared with urinefrom a female carrying a male fetus. When a urine sample from a pregnantfemale carrying a male fetus contacts PTA, PTA remains intact in theurine and forms insoluble complexes with urinary proteins andglycoproteins. Thus, this urine sample appears as a white colloidalsuspension in the PTA assay for at least about 12 hours.

In contrast, for urine from a pregnant female carrying a female fetus,although PTA initially forms insoluble complexes with urinary proteinsand glycoproteins, the complexes gradually decrease as PTA is beingreduced by the urine. The reaction mixture becomes transparent overtime. The time period for the reaction mixture to turn from a whitecolloidal suspension to clear solution may vary among different urinesamples. In some embodiments, the time period may range from about 5minutes to about 12 hours. Shorter or longer time periods may also beappropriate, such as about 1 minute, 2 minutes, 3 minutes, 4 minutes, 13hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours and 20hours.

Tungsten or molybdenum is preferred as the addenda atom of theheteropoly acid or its salts used in the present invention. A heteropolyacid or its salts may consist of one or more than one type of addendaatoms. The combinations of the addenda atoms include molybdenum andtungsten, vanadium and molybdenum, and vanadium, molybdenum andtungsten. The hetero atom of a heteropoly acid or its salts is selectedfrom the group consisting of phosphorus, arsenic, silicon and germanium.Phosphorus is preferred as the hetero atom. Heteropoly acids or theirsalts comprising molybdenum and phosphorus, molybdenum and arsenic, ortungsten and phosphorus are preferred embodiments. A heteropoly acid orits salts may be selected from the group consisting ofphospho-12-molybdic acid, phospho-18-molybdic acid, 12-molybdoarsenicacid, 18-molybdoarsenic acid, 11-molybdo-l-vanadophosphoric acid,10-molybdo-2-vanadophosphoric acid, phospho-12-tungstic acid,phospho-18-tungstic acid, phospho-24-tungstic acid and9-molybdo-3-vanadophosphoric acid. Phospho-18-molybdic acid,phospho-12-tungstic acid, phospho-18-tungstic acid andphospho-24-tungstic acid are preferred embodiments. Heteropoly acids andtheir salts may be used individually or their combinations may be used.

An excellent correlation (R²>0.99) was reported between total phenoliccontents as measured by Folin-Ciocalteau reagent(Phosphomolybdic-Phosphotungstic Acid Reagents) and antioxidant activityas measured by FRAP, TEAC or DPPH (1,1-Diphenyl-2-Picrylhydrazyl)assays. Connor et. al. Changes in fruit antioxidant activity amongblueberry cultivars during cold temperature storage. J. Aaric. FoodChem. 2002, (50), 893-989. Because these assays are based on similarredox reactions, it is important to use one assay that is commonlyaccepted and validated. The total phenols assay by Folin-Ciocalteaureagent is conducted at basic conditions (e.g., pH 10). The PTA test mayalso be performed at a basic pH. Since each of the three main estrogens(i.e., estradiol, estriol, estrone) has a phenol group which contributesto their antioxidant properties, reduction of heteropoly acids has beenseen as a measure of both the phenolic contents and antioxidantproperties. Therefore, under these conditions, the phosphotungsticreducing capacity correlates with radical scavenging capacity ofestrogens in processed urine sample.

The overall redox activity of a body fluid may be assayed by achromogenic chemical comprising an oxidation-reduction sensitivemetallic ion. The metallic ion may be selected from the group consistingof copper, iron, chromium, tungsten, molybdenum ions in an oxidizedstate.

The chromogenic chemical may be a metal-organic complex which, due toits extremely high oxidizing activity (correlated with high redoxpotential), can be reduced by many reducing agents and undergo change incolor. The ferric reducing/antioxidant power (FRAP) assay can be used toassay the overall reducing activity of a body fluid. The ferrictripyridyltriazine (Fe(III)-TPTZ) complex is reduced under acidicconditions by a reducing agent to form ferrous tripyridyltriazine(Fe(II)-TPTZ). Fe(III)-TPTZ is yellow in color. The production ofFe(II)-TPTZ is easily detectable and measurable because of its intenseblue color with a maximum absorption at wavelength 593 nm.

The FRAP assay may be conducted under harsh conditions, where the urinefrom a female carrying a male fetus will have a comparatively higheroverall reducing activity than urine from a female carrying a femalefetus. Accordingly, a urine sample from a female carrying a male fetusis able to reduce Fe(III) to Fe(II), or Fe(III)-TPTZ to Fe(II)-TPTZ,with the reaction mixture turning dark blue. In contrast, a urine samplefrom a female carrying a female fetus is not able to reduce Fe(III), orFe(III)-TPTZ, and thus, the reaction mixture remains yellow.

Potassium ferricyanide may also be used in the FRAP assay. Oyaizu.Studies on products of Browning reaction. J. Nutrition. 44: 307-315(1986). Under the harsh conditions of the FRAP assay, the urine from afemale carrying a male fetus will have comparatively higher overallreducing activity than urine from a female carrying a female fetus.Accordingly, a urine sample from a female carrying a male fetus is ableto reduce potassium ferricyanide to potassium ferrocyanide, which isthen able to form dark blue product when ferric chloride is added. Incontrast, a urine sample from a female carrying a female fetus is notable to reduce potassium ferricyanide, and, thus, the reaction mixtureremains golden yellow or faint green in color.

For fraternal multiples with different genders, when the FRAP assay isconducted, the urine with a comparatively higher overall reducingactivity than urine from a female carrying a female fetus correlateswith at least one male fetus, whereas the urine with comparable overallreducing activity to urine from a female carrying a female fetuscorrelates with at least two female fetuses.

The overall reducing activity of a body fluid may be assayed by thereduction of Cu²⁺ to Cu⁺, which may be detected by the complex formationbetween Cu⁺ and bathocuproine. The amount of Cu⁺ being generatedcorrelates with the overall reducing activity of the body fluid. TheCu⁺-bathocuproine complex is stable and has a maximum absorption atwavelength around 480-490 nm. In one embodiment, the amount of Cu⁺ beinggenerated is quantified by referring to a standard curve, which usesuric acid as the reducing agent.

The overall reducing or antioxidant activity of the body fluid may beassayed by its ability to capture a free radical in the presence of aredox indicator. In one embodiment, the radical is hydroxy radical (OH.)generated either by a hydroxyl radical-generating system or internallywithin the body fluid, with sodium salicylate as the redox indicator. Inthe absence of other antioxidant (or reducing agent), the colorlesssalicylate ion reacts with OH. to produce dihydroxybenzoic acid, whichis blue in color and has a maximum absorption at wavelength 510 nm. Whenother antioxidant (or reducing agent) is present, hydroxyl radical issequestered or trapped by this antioxidant (or reducing agent) instead.As a result, sodium salicylate stays intact and the reaction mixtureremains colorless.

In another embodiment, the overall redox activity of the body fluid maybe assayed by its ability to facilitate the generation of free radicals.The assay reagents may comprise sodium salicylate and a hydroxyl radicalgenerating system including a ferric compound, such as ferric sulfate,and H₂O₂. When the reducing agent is present, it reduces ferric toferrous, which results in further reduction of H₂O₂ to generate hydroxylradical OH. through the Fenton reaction. Salicylate ion then reacts withOH. to produce dihydroxybenzoic acid that is blue in color. The reactionis diagramed below.

Fe³⁺+Reducing Agent→Fe²⁺Oxidized Reducing Agent

Fe²⁺+H₂O₂→Fe³⁺+OH.+OH—

Sodium Salicylate+2OH.→Dihydroxybenzoic Acid

In the absence of the reducing agent, no OH. is generated. As aconsequence, sodium salicylate remains colorless.

There are various ways to generate radicals. For example, In addition toFenton reactions, hydroxyl radicals can be generated by a) ultrasound(Free radical generation by ultrasound in aqueous and nonaqueoussolutions. P. Riesz et al. Env. Health Perspectives 1985 (64) 233-252);b) xanthine/xanthine oxidase system (Generation of hydroxyl radical byenzymes, chemicals, and human phagocytes in vitro. John E. Repine et.al. J. Clin. Invest. 1979 (64) 1642-1651); c) electron paramagneticresonance (Hydroxyl radical generation by electron paramagneticresonance as a new method to monitor ambient particulate mattercomposition. Tingming Shi et. al. J. Environ. 2003, (5), 550-556); d)phagocytic activity of human leukocytes (human granulocyte generation ofhydroxyl radical. Stephen J. Weiss et. al. J. Exp. Med. 1978 (147)316-323); e) oxidation of certain chemicals (Enhanced generation ofhydroxyl radical and sulfur trioxide anion radical from oxidation ofsodium sulfite, nickel (II) sulfite, and nickel subsulfide in thepresence of nickel (II) complexes. Xianglin Shi et. al. Environ. HealthPerspectives 1994, 102(3)); f) photolysis of N-hydroxypyridinethiones(Photolysis of N-hydroxypyridinethiones: a new source of hydroxylradicals. Bernd Epe et. al. Nucleic Acids Res. 1996, 24(9), 1625-1631);g) UV-induced photolysis of hydrogen peroxide and UV irradiation ofparticles of titanium dioxide, etc.

Additionally, hydroxyl radicals can be produced internally throughcontribution of iron from hemoglobin and its metabolites in urine in thepresence of H202. Accordingly, hemoglobin has been regarded as aninternal Fenton reagent. In the presence of a superoxideanion-generating system (e.g., hypoxanthine and xanthine oxidase),haemoglobin promotes hydroxyl radical formation in a dose dependentfashion. Hydroxyl radical may be generated primarily via reactionbetween the ferrous heme iron and H₂O₂. (Hemoglobin: A biologic FentonReagent. Sayed M. H. Sadrzadeh et. al. J. Biol. Chem. 1984, 259(23),14354-14356). The H₂O₂decomposes heme and its metabolites, and releasesiron from the protein (Formation of hydroxyl radicals from hydrogenperoxide in the presence of iron. Is haemoglobin a biological Fentonreagent? Alain Puppo et. al., Biochem J. 1988 (249), 185-190.)

The applicant has observed that urine samples from women with femalegender related characteristics (e.g., a pregnant woman carrying a femalefetus) have higher porphyrin content than that of women with male genderrelated characteristics (e.g., a pregnant woman carrying a male fetus).This may be due to relatively higher degradation of heme moleculesoccurring in-vivo in women with female gender related characteristics.

The present invention provides a method of determining the gender of anunborn child comprising the steps of: (a) contacting a body fluid from apregnant female with free radicals; (b) measuring free radicalscavenging activity of the body fluid; (c) comparing the free radicalscavenging activity of the body fluid with at least one standard; and(d) determining the gender of the unborn child.

The free radical scavenging activity may be measured by methylene blue.Methylene blue may be in a solid form, in a solution, or attached to asolid surface. In one embodiment, the gender of an unborn child isdetermined to be female if there is substantially no decolorization ofmethylene blue; the gender of an unborn child is determined to be maleif there is decolorization of methylene blue within about 1 hour.

The free radicals may be DPPH (1,1-Diphenyl-2-Picrylhydrazyl) radicals,or galvinoxyl radical, i.e., GV(2,6-di-tert-butyl-α-(3,5-di-tert-butyl-oxo-2,5-cyclohexanedien-1-ylidene)-p-tolyloxy)radical. DPPH may be in a solid form, in a solution, or attached to asolid surface. The free radicals may be hydroxyl radicals or superoxideradicals. The hydroxyl radicals may be generated internally orexternally using heat, ultrasound, irradiation, photolysis, electronparamagnetic resonance, electrolysis, chemical, electrochemical orenzymatic means. The hydroxyl radicals may be generated by the Fentonreaction (see above for the reaction scheme). The Fenton reaction mayuse iron ions, copper ions or titanium dioxide as catalysts.

In one specific example, when one drop (or two to three drops) of aprocessed urine sample from a non-pregnant or pregnant female is addedto 1 ml (or 0.5 ml) of 100 ppm solution of DPPH radical in methanol (orin acetonitrile), the rate of decolorization of the DPPH solution isfaster in samples having female gender related characteristics (e.g., aurine sample from a pregnant woman carrying a female fetus) compared tosamples having male gender related characteristics (e.g., a urine samplefrom a pregnant woman carrying a male fetus). Specifically, when therate of disappearance of DPPH's purple color is measured using aspectrophotometer at 517 nm, the area under curve (AUC) for a samplehaving female gender related characteristics is significantly lower thanthat of a sample having male gender related characteristics. Thedifference may be observed with the naked eye. Whereas a sample havingfemale gender related characteristics may completely decolorize within 2minutes, a sample having male gender related characteristics may takemuch longer time (e.g., from about 1 hour to about 12 hours) or evennever completely decolorize in several days. The length of time for DPPHto decolorize may vary depending on factors such as the properties ofthe DPPH reagent used, the concentration of the DPPH solution used, theestrogens in the sample, etc.

Estrogens may be the only radical scavenging components in an aged urinesample. Therefore, a urine sample having female gender relatedcharacteristics will have higher radical scavenging activity as comparedto a urine sample having male gender related characteristics. Inclusionof additional radicals, like DPPH, induces competition between hydroxylradicals and DPPH radicals. By modifying urine processing protocol, itis possible to suppress or promote the generation of hydroxyl radicals.When hydroxyl radical generation is diminished or blocked, estrogensscavenge DPPH radicals instead and a sample having female gender relatedcharacteristics will decolorize relatively much faster than a samplehaving male gender related characteristics. A sample having femalegender related characteristics may decolorize in less than about 1minute, or less than about 5 seconds, whereas a sample having malegender related characteristics may decolorize after about 1 hour, afterabout 2 hours, after about 3 hours or even longer.

On the other hand, when the sample processing conditions are such thathydroxyl radical production is promoted or retained, there iscompetition between DPPH radicals and hydroxyl radicals to react withestrogens. The unused DPPH radicals appear to preferably be reduced byan unused portion of hydroxyl radicals. Under such conditions,decolorization of DPPH is faster in a sample having male gender relatedcharacteristics than that of a sample having female gender relatedcharacteristics. A sample having male gender related characteristics maydecolorize in about 15 minutes, whereas a sample having female genderrelated characteristics may take longer to decolorize, for example,about 1 hour to about 24 hours, about 1 hour to about 12 hours, about 1hour, about 12 hours, or about 24 hours. Ultimately both types ofsamples will decolorize from purple to yellow. Due to variability of sexhormone levels in samples, it is relatively difficult to clearlyevaluate the gender related characteristics under these conditions, and,as a result, the error rate may be high.

Therefore, when DPPH radical is being used, a preferable embodimentwould be to block the hydroxyl radical generation to get an accurateevaluation of fetal gender related characteristics.

The overall redox activity of the body fluid may also be detected usingredox sensitive polymers such as polyaniline. Polyaniline is polymerizedfrom the aniline monomer and has the following structure,

where x is half the degree of polymerization. Polyaniline can be foundin one of three oxidation states. The leucoemeraldine form ofpolyaniline (n=1, m=0) is the fully reduced state and is white orcolorless. Pernigraniline (n=0, m=1) is the fully oxidized state and isblue or violet. The emeraldine (n=m=0.5) form of polyaniline is theintermediate state and is green or blue. The color change associatedwith polyaniline in different oxidation states may be exploited to usepolyaniline as a redox indicator. Huanga et al. Development andcharacterization of flexible electrochromic devices based on polyanilineand poly(3,4-ethylenedioxythiophene)-poly(styrene sulfonic acid).Electrochimica Acta. 51, 26: 5858-5863 (2006).

In another embodiment, the overall reducing activity of a body fluid maybe assayed by an in situ methodology. For example, a redox indicator,which may be selected from the group consisting of thionine, toluidineblue O and cresyl violet, is immobilized to agarose beads. The agarosebeads are then packed into a flow cell. The body fluid is allowed topass through the flow cell and the redox states of the redox indicatoris monitored by spectrophotometry. Jones. Evaluation of immobilizedredox indicators as reversible, in situ redox sensors for determiningFe(III)-reducing conditions in environmental samples. Talanta. 2001. 55,4: 699-714.

Voltammetry may be used in the present invention. Many studies have usedvoltammetry to assay antioxidant activities. (Chavion S. et al. The useof cyclic voltammetry for the evaluation of the antioxidant capacity.Free Radic. Biol. Med. 2000, 28, 860-870). Voltammetric assays are basedon cyclic voltammetry (CV), square wave voltammetry (SWV), anddifferential pulse voltammetry (DPV). Antioxidants are oxidized under atypical voltage which results in a faradaic current proportional to theconcentration of antioxidants (Supalkova V. et al. Electroanalysis ofplant thiols. Sensors. 2007, 7, 932-959). In assaying antioxidants inbiological matrices, a close correlation was found for the SWV resultsand the FRAP data. One of the advantages of SWV is the ability todistinguish two basic types of low molecular weight antioxidants, i.e.,ascorbic/uric acids and thiol-containing molecules. SWV can be conductedwithin a relatively short period of time with or without prior samplemodification. (Miroslav P. et al., Ferric reducing antioxidant power andsquare wave voltammetry for assay of low molecular weight antioxidantsin blood plasma. Sensors 2009, (9), 9094-9103).

The overall redox activity of a body fluid may be detected by generalreducing sugar tests using copper ion, neocuproine or alkalineferricyanide method with or without color formation witho-phenanthroline complex. Prado et al. Phytochem. Analysis. 9 (2): 58-62(1998).

The overall redox activity of the body fluid may also be detected by theOxygen Radical Absorbance Capacity (ORAC) assay, which measures bothlipophilic and hydrophilic antioxidant capacity using the same peroxylradical generator. The ORAC assay measures the oxidative degradation ofa fluorescent molecule (such as beta-phycoerythrin or fluorescin) afterbeing mixed with free radical generators such as azo-initiatorcompounds. Azo-initiator compounds produce peroxyl free radical, whichdamages the fluorescent molecule resulting in loss of fluorescence.Antioxidant or reducing agent such as estrogen is able to protect thefluorescent molecule from the oxidative degeneration. The degree ofprotection by the antioxidant or reducing agent may be quantified usinga fluorometer. Williams et al. A comparison of mammalian and plantestrogens on vascular reactivity in young and old mice with or withoutdisruption of estrogen receptors. Current Topics in Nutraceutical Res.2, 4: 191 (2004).

The present invention provides a method of determining the gender of anunborn child comprising the steps of: (a) performing oxygen radicalabsorbance capacity (ORAC) assay on a body fluid from a pregnant female;(b) calculating the ORAC value; and, (c) comparing the ORAC value fromstep (b) with at least one standard to determine the gender of theunborn child.

The present invention further provides a method of determining thegender of an unborn child comprising the steps of: (a) performinghydroxyl radical antioxidant capacity (HORAC) assay on a body fluid froma pregnant female; (b) calculating the HORAC value; (c) comparing theHORAC value from step (b) with at least one standard; and (d)determining the gender of the unborn child.

The HORAC assay is based on the oxidation of fluorescein by hydroxylradicals via a classic hydrogen atom transfer (HAT) mechanism. Freeradicals are generated by hydrogen peroxide (H₂O₂). The hydroxylradicals thus generated quench the fluorescence of fluorescein overtime. The antioxidants block the hydroxyl radical mediated oxidation offluorescein until all of the antioxidant activity in the sample isexhausted, after which the H₂O₂ radicals react with and quench thefluorescence of fluorescein. The area under the fluorescence decay curve(AUC) is used to quantify the total hydroxyl radical antioxidantactivity in a sample and is compared to a standard curve obtained usingvarious concentrations of gallic acid. The fluorescent HORAC assayprovides a direct measurement of antioxidant capacity againsthydrophilic chain-breaking hydroxyl radicals.

When fluorescein is used as radical quenching chemical, the gender ofthe unborn child is determined to be male if the HORAC value from step(b) is less than about 5000; the gender of the unborn child isdetermined to be female if the value from step (b) is greater than about6000. When other compounds (such as methylene blue) are used as radicalquenching chemicals, the HORAC value from step (b) may have differentrange for male and/or female unborn child.

In one embodiment, there is provided a method of determining the genderof an unborn child comprising the steps of: (a) performing hydroxylradical antioxidant capacity (HORAC) assay on a body fluid from apregnant female; (b) calculating the HORAC value; and (c) determiningthe gender of the unborn child to be male if the HORAC value from step(b) is less than about 5000, or to be female if the value from step (b)is greater than about 6000.

The reaction of a body fluid sample with methylene blue is similar tothe HORAC test where a sample associated with female gender relatedcharacteristics (e.g., a urine sample from a pregnant woman carrying afemale fetus) exhibits greater radical scavenging capacity. In aspecific example, a hydroxyl radical generation system is provided withmethylene blue being coated on a solid support (e.g., a piece of paper)and used as a hydroxyl radical probe. A urine sample from a femaleexhibiting male gender related characteristics (e.g., a pregnant womancarrying a male fetus) would decolorize methylene blue, forming a bluering with a white center, whereas methylene blue mixed with a urinesample from a female exhibiting female gender related characteristics(e.g., a pregnant woman carrying a female fetus) would remain blue(e.g., no decolorization). Without being limited to any specificphysiological mechanism, it is believed that in samples with femalegender related characteristics, the hydroxyl radicals preferentiallyreact with relatively higher amount of estrogens in the urine sample anddo not decolorize methylene blue. In contrast, in samples exhibitingmale gender related characteristics, there are less estrogens to absorbhydroxyl radicals, therefore leaving enough hydroxyl radicals todecolorize methylene blue. The rate of decolorization depends upon thetype of urine sample as well as on the processing method.

Any suitable assays similar to the HORAC or ORAC assays can be used inthe present invention. For these assays, any suitable free radicalgenerators, and/or any suitable fluorescent molecules can be used.

The overall redox activity of the body fluid may be detected by theAntioxidant Capacity (AOC) assay using the3-ethylbenzothiazoline-6-sulfonate (ABTS) system or TPTZ. Bahramikia etal. A comparison of Antioxidant Capacities of ethanol extracts of S.Hortensis and A. Dracunculus leaves. Pharmacology Online 2: 694-704(2008).

The overall redox activity of the body fluid may be detected by the useof immobilized chromogenic radicals. In one embodiment, either of twostable lipophylic chromogenic radicals, DPPH(2,2-diphenyl-1-picrylhydrazyl) radical and GV(2,6-di-tert-butyl-α-(3,5-di-tert-butyl-oxo-2,5-cyclohexanedien-1-ylidene)-p-tolyloxy)radical, is immobilized in plasticized polymer films, such as polyvinylchloride (PVC) films. When the polymer film containing immobilized2,2-diphenyl-1-picrylhydrazyl (DPPH) radical is reduced by a body fluid,it changes color irreversibly from purple (maximum absorption atwavelength 520 nm) to yellow. Steinberg et al. Chromogenic radical basedoptical sensor membrane for screening of antioxidant activity. Talanta8: 15 (2006).

The overall redox activity of the body fluid may be detected byimmunoassay such as immunofluorescence, using antibodies against genderrelated molecules; chemiluminescence; bioluminescence such as greenfluorescent proteins; antibody-tagged gold nanoparticles of differingsizes; chemical tagging or fluorescent dyes. Kohen et al. Recentadvances in chemiluminescence-based immunoassays for steroid hormones.J. Steroid Biochem. 27, 1-3: 71-79 (1987).

The overall oxidizing activity of the body fluid may be assayed byadding a source of ferrous ions to the body fluid, whereby oxidants inthe sample oxidize at least a portion of the ferrous ions to ferricions. Then a chromogenic compound is added to the sample, which reactswith at least a portion of the ferric ions. U.S. Patent Publication No.20040259186.

The overall reducing activity of the body fluid may be assayed usingreagents selected from the group consisting of ferricyanide salts,dichromate salts, permanganate salts, vanadium oxides,dichlorophenolindophenol, osmium bipyridine complexes, and quinones. Theoverall oxidizing activity of the body fluid may be assayed usingreagent selected from the group consisting of iodine, triiodide salts,ferrocyanide salts, ferrocene, [Cu(NH3)₄]²⁺ salts and [Co(NH3)₆]³⁺ Gsalts. The redox indicator of the present methods may be selected fromthe group consisting of 2,6-dichlorophenolindophenol (DCPIP),3,3′,5,5′tetramethylebenzidine (TMB), 1,4-phenylenediamine (DMPDA),Phenanthridine, 2,6-Dichloroindophenol,2,2′-Azino-bis(3-ethylbenzthiazoline-6-sulfonic) acid,N,N-Dimethylphenylenediamine, 2-Amino-p-cresol (APC), Xylenol orange,8-Hydroxy-7-iodo-5-quinoline-sulfonic acid and4,5-Dihydroxy-1,3-benzene-di-sulfonic acid.

The present invention provides a method of determining the gender of anunborn child comprising the steps of: (a) performing the HORAC assay ona body fluid from a pregnant female; (b) measuring levels oftestosterone (T) and estrogens (E) in the body fluid, wherein step (b)can be performed before or after step (a); (c) calculating the value ofHORAC/(E/T), wherein E/T is the ratio of the level of testosterone tothe level of estrogens, and wherein the value of HORAC/(E/T) is theratio of the HORAC value to the E/T ratio; and, (d) determining thegender of the unborn child to be male if the value of HORAC/(ET) fromstep (c) is less than about 150, or to be female if the value from step(c) is greater than about 200.

Also provided in the present invention is a method of determining thegender of an unborn child comprising the steps of: (a) performing theHORAC assay on a body fluid from a pregnant female; (b) measuring levelsof testosterone (T), estrogens (E), progesterone (P) and gonadotropins(G) in the body fluid, wherein step (b) can be performed before or afterstep (a); (c) calculating the value of HORAC/[E−(T+P+G)], wherein[E−(T+P+G)] is the difference between the level of estrogens (E) and thetotal levels of testosterone (T), progesterone (P) and gonadotropins(G), and wherein the value of HORAC/[E−(T+P+G)] is the ratio of theHORAC value to the [E−(T+P+G)] value; (d) comparing theHORAC/[E−(T+P+G)] value from step (c) with at least one standard; and(e) determining the gender of the unborn child.

The present invention further provides a method of determining thegender of an unborn child comprising the steps of: (a) performing theHORAC assay on a body fluid from a pregnant female; (b) measuring levelsof testosterone (T), estrogens (E), progesterone (P) and gonadotropins(G) in the body fluid, wherein step (b) can be performed before or afterstep (a); (c) calculating the value of HORAC/[(E+T)/(P+G)] wherein[(E+T)/(P+G)] is the ratio of the total levels of estrogens (E) andtestosterone (T) to the total levels of progesterone (P) andgonadotropins (G), and wherein the value of HORAC/[(E+T)/(P+G)] is theratio of the HORAC value to the [(E+T)/(P+G)] value; (d) comparing theHORAC/[(E+T)/(P+G)] value from step (c) with at least one standard; and(e) determining the gender of the unborn child.

The present invention provides a method of determining the gender of anunborn child comprising the steps of: (a) contacting a body fluid from apregnant female with a solid surface having antibodies specific toestrogens, antibodies specific to testosterone, antibodies specific toprogesterone and antibodies specific to hCG-beta; (b) contacting thesolid surface from step (a) with at least one redox indicator; (c)measuring redox activity of the solid surface; (d) comparing the redoxactivity of the solid surface with at least one standard; and (e)determining the gender of the unborn child. Sex hormones mayalternatively be released from the solid surface before the redoxactivity of the sex hormones is measured.

The redox activity may be measured by Ferric reducing/antioxidant power(FRAP) assay, and/or Ferric reducing/antioxidant power assay usingferric tripyridyltriazine (FRAP-TPTZ).

The present invention also provides methods for pre-conception babygender planning by assaying the overall redox activity and or radicalscavenging activity of a female's urine or other body fluid. The bodyfluid may be obtained anytime during a menstrual cycle, but preferablyaround the time of ovulation.

Without being limited to any specific physiological mechanism, it isbelieved that ovulation generates at least two types of uterineenvironments during alternate menstrual cycles, presumably due to therelease of two different types of ova. One type of ovum is associatedwith uterine environment to be compatible with fertilization by a Ychromosome-bearing sperm to produce a male fetus, whereas the other typeof ovum is associated with uterine environment to be compatible withfertilization by an X chromosome-bearing sperm to produce a femalefetus. The overall redox activity of the maternal urine correlates withthe gender specific compatibility of the ovum released in a particularmenstrual cycle.

Under harsh assay conditions or with aging, the urine sample of anon-pregnant female carrying an ovum compatible with fertilization by aY chromosome-bearing sperm will have comparatively higher reducingactivity than urine from a non-pregnant female carrying an ovumcompatible with fertilization by an X chromosome-bearing sperm (seeTable 1 for summary) It is believed that a female produces two types ofova which generally alternate every month, i.e., if in one month theovum being produced is compatible with fertilization by an Xchromosome-bearing sperm, then in the next month the ovum being producedis compatible with fertilization by a Y chromosome-bearing sperm. Thepre-conception urine assay can help a couple conceive a baby having adesired gender. For example, if a couple desires a baby girl, it isrecommended that they plan to conceive in any month when the ovum beingproduced is compatible with fertilization by an X chromosome-bearingsperm. Similarly, if a couple wishes a baby boy, it is recommended thatthey plan to conceive in any month when the ovum being produced iscompatible with fertilization by a Y chromosome-bearing sperm. Thepurpose of the present pre-conception methods is to increase the chancesof conceiving a baby of the desired gender by advising appropriatemenstrual cycles for conception.

The present invention provides a method for pre-conception baby genderplanning comprising the steps of: (a) contacting a body fluid from anon-pregnant female with at least one redox indicator; (b) measuringredox activity of the body fluid; and, (c) comparing the redox activityof the body fluid with at least one standard to determinegender-specific compatibility of the ovum released in a menstrual cycle.The body fluid may be obtained near ovulation or middle of the menstrualcycle. The body fluid may be processed prior to step (a).

If two urine samples collected in two consecutive months manifestdifferent redox activities, it suggests that the female has a regularalternating ovulation pattern as described above. If both urine samplesdemonstrate comparatively higher reducing activity than urine from anon-pregnant female carrying an ovum compatible with fertilization by anX chromosome-bearing sperm, the female may have an irregular ovulationpattern which is difficult to normalize according to the presentinvention. If both urine samples demonstrate comparatively higheroxidizing activity than urine from a non-pregnant female carrying anovum compatible with fertilization by a Y chromosome-bearing sperm, thefemale's irregular ovulation pattern may be normalized through diet andexercise.

As described above, any suitable assay method, or a combination of twoor more such assay methods, capable of measuring the overall redoxactivity of the body fluid may be used with the present pre-conceptionassay or the present post-conception assay.

The overall redox activity of the body fluid during a menstrual cyclewithin which a female conceives is continued and maintained throughoutpregnancy. Namely, if her pre-conception body fluid has a comparativelyhigher overall reducing activity than that of the body fluid from anon-pregnant female carrying an ovum compatible with fertilization by anX chromosome-bearing sperm under the harsh condition (for example, inthe FRAP assay), her ovum is compatible with fertilization by a Ychromosome-bearing sperm. Consistently, her post-conception urine samplewould also have a higher overall reducing activity than that of urinefrom a female carrying a female fetus, which is correlated with a malefetus. Conversely, if her pre-conception urine sample has a higheroverall oxidizing activity than that of urine from a non-pregnant femalecarrying an ovum compatible with fertilization by a Y chromosome-bearingsperm under harsh conditions, her ovum is compatible with fertilizationby an X chromosome-bearing sperm. Likewise, her post-conception urinesample would also have a higher overall oxidizing activity than that ofurine from a female carrying a male fetus, which is correlated with afemale fetus.

Provided in the present invention is a method of determining genderspecific compatibility of an ovum released in a menstrual cycle forpre-conception baby gender planning comprising the steps of: (a)measuring levels of testosterone (T), estrogens (E), progesterone (P)and gonadotropins (G) in a body fluid from a non-pregnant female; (b)calculating [E−(T+P+G)] as the difference between the level of estrogens(E) and the total levels of testosterone (T), progesterone (P) andgonadotropins (G); (c) comparing the [E−(T+P+G)] value from step (b)with at least one standard; and (d) determining the gender specificcompatibility of the ovum released in the menstrual cycle.

In one embodiment, there is provided a method of determining genderspecific compatibility of an ovum released in a menstrual cycle forpre-conception baby gender planning comprising the steps of: (a)measuring levels of testosterone (T), estrogens (E), progesterone (P)and gonadotropins (G) in a body fluid from a non-pregnant female; (b)calculating [E−(T+P+G)] as the difference between the level of estrogens(E) and the total levels of testosterone (T), progesterone (P) andgonadotropins (G); and (c) determining the gender specific compatibilityof the ovum released in the menstrual cycle to be male if the[E−(T+P+G)] value from step (b) ranges from about 300 to about 1,500, orto be female if the [E−(T+P+G)] value from step (b) ranges from about−10 to about −800.

The present invention provides a method of determining gender specificcompatibility of an ovum released in a menstrual cycle forpre-conception baby gender planning comprising the steps of: (a)measuring levels of testosterone (T), estrogens (E), progesterone (P)and gonadotropins (G) in a body fluid from a non-pregnant female; (b)calculating (E+T)/(P+G) as the ratio of the total levels of estrogens(E) and testosterone (T) to the total levels of progesterone (P) andgonadotropins (G); (c) comparing the ratio (E+T)/(P+G) from step (b)with at least one standard; and (d) determining the gender specificcompatibility of the ovum released in the menstrual cycle.

In one embodiment, there is provided a method of determining genderspecific compatibility of an ovum released in a menstrual cycle forpre-conception baby gender planning comprising the steps of: (a)measuring levels of testosterone (T), estrogens (E), progesterone (P)and gonadotropins (G) in a body fluid from a non-pregnant female; (b)calculating (E+T)/(P+G) as the ratio of the total levels of estrogens(E) and testosterone (T) to the total levels of progesterone (P) andgonadotropins (G); and (c) determining the gender specific compatibilityof the ovum released in the menstrual cycle to be male if the(E+T)/(P+G) ratio from step (b) is greater than about 1.4, or to befemale if the (E+T)/(P+G) ratio from step (b) is less than about 1.2.

The present invention also provides a method of determining genderspecific compatibility of an ovum released in a menstrual cycle forpre-conception baby gender planning comprising the steps of: (a)performing hydroxyl radical antioxidant capacity (HORAC) assay on a bodyfluid from a non-pregnant female; (b) calculating the HORAC value; (c)comparing the HORAC value from step (b) with at least one standard; and(d) determining the gender specific compatibility of the ovum releasedin the menstrual cycle.

In one embodiment, there is provided a method of determining genderspecific compatibility of an ovum released in a menstrual cycle forpre-conception baby gender planning comprising the steps of: (a)performing hydroxyl radical antioxidant capacity (HORAC) assay on a bodyfluid from a non-pregnant female; (b) calculating the HORAC value; and(c) determining the gender specific compatibility of the ovum releasedin the menstrual cycle to be male if the HORAC value from step (b) isless than about 5000, or to be female if the value from step (b) isgreater than about 6000.

The present invention further provides a method of determining genderspecific compatibility of an ovum released in a menstrual cycle forpre-conception baby gender planning comprising the steps of: (a)performing hydroxyl radical antioxidant capacity (HORAC) assay on a bodyfluid from a non-pregnant female; (b) measuring levels of testosterone(T) and estrogens (E) in the body fluid, wherein step (b) can beperformed before or after step (a); (c) calculating the value ofHORAC/(E/T), wherein E/T is the ratio of the level of testosterone tothe level of estrogens, and wherein the value of HORAC/(E/T) is theratio of the HORAC value to the E/T ratio; (d) comparing the ratioHORAC/(E/T) from step (c) with at least one standard; and (e)determining the gender specific compatibility of the ovum released inthe menstrual cycle.

In one embodiment, there is provided a method of determining genderspecific compatibility of an ovum released in a menstrual cycle forpre-conception baby gender planning comprising the steps of: (a)performing hydroxyl radical antioxidant capacity (HORAC) assay on a bodyfluid from a non-pregnant female; (b) measuring levels of testosterone(T) and estrogens (E) in the body fluid, wherein step (b) can beperformed before or after step (a); (c) calculating the value ofHORAC/(E/T), wherein E/T is the ratio of the level of testosterone tothe level of estrogens, and wherein the value of HORAC/(E/T) is theratio of the HORAC value to the Elf ratio; and (d) determining thegender specific compatibility of the ovum released in the menstrualcycle to be male if the value of HORAC/(ET) from step (c) is less thanabout 150, or to be female if the value from step (c) is greater thanabout 200.

The present invention provides a method of determining gender specificcompatibility of an ovum released in a menstrual cycle forpre-conception baby gender planning comprising the steps of: (a)performing hydroxyl radical antioxidant capacity (HORAC) assay on a bodyfluid from a non-pregnant female; (b) measuring levels of testosterone(T), estrogens (E), progesterone (P) and gonadotropins (G) in the bodyfluid, wherein step (b) can be performed before or after step (a); (c)calculating the value of HORAC/[E−(T+P+G)], wherein [E−(T+P+G)] is thedifference between the level of estrogens (E) and the total levels oftestosterone (T), progesterone (P) and gonadotropins (G), and whereinthe value of HORAC/[E−(T+P+G)] is the ratio of the HORAC value to the[E−(T+P+G)] value; (d) comparing the HORAC/[E−(T+P+G)] value from step(c) with at least one standard; and (e) determining the gender specificcompatibility of the ovum released in the menstrual cycle to be male ifthe HORAC/[E−(T+P+G)] value from step (c) falls within a range of thestandard with a male gender specific compatibility, or to be female ifthe HORAC/[E−(T+P+G)] value from step (c) falls within a range of thestandard with a female gender specific compatibility.

The present invention provides a method of determining gender specificcompatibility of an ovum released in a menstrual cycle forpre-conception baby gender planning comprising the steps of: (a)performing hydroxyl radical antioxidant capacity (HORAC) assay on a bodyfluid from a non-pregnant female; (b) measuring levels of testosterone(T), estrogens (E), progesterone (P) and gonadotropins (G) in the bodyfluid, wherein step (b) can be performed before or after step (a); (c)calculating the value of HORAC/[(E+T)/(P+G)] wherein [(E+T)/(P+G)] isthe ratio of the total levels of estrogens (E) and testosterone (T) tothe total levels of progesterone (P) and gonadotropins (G), and whereinthe value of HORAC/[(E+T)/(P+G)] is the ratio of the HORAC value to the[(E+T)/(P+G)] value; (d) comparing the HORAC/[(E+T)/(P+G)] value fromstep (c) with at least one standard; and (e) determining the genderspecific compatibility of the ovum released in the menstrual cycle to bemale if the HORAC/[(E+T)/(P+G)] value from step (c) falls within a rangeof the standard with a male gender specific compatibility, or to befemale if the HORAC/[(E+T)/(P+G)] value from step (c) falls within arange of the standard with a female gender specific compatibility.

Also provided in the present invention method of determining genderspecific compatibility of an ovum released in a menstrual cycle forpre-conception baby gender planning comprising the steps of: (a)contacting a body fluid from a non-pregnant female with free radicals;(b) measuring free radical scavenging activity of the body fluid; (c)comparing the free radical scavenging activity of the body fluid with atleast one standard; and (d) determining gender specific compatibility ofthe ovum released in a menstrual cycle.

The free radicals may be DPPH (1,1-Diphenyl-2-Picrylhydrazyl) radical orGV(2,6-di-tert-butyl-α-(3,5-di-tert-butyl-oxo-2,5-cyclohexadien-1-ylidene)-p-tolyloxyradical.

When DPPH is used, in one embodiment, the gender specific compatibilityof the ovum is determined to be female if decolorization of DPPH occurswithin about 1 minute, whereas the gender specific compatibility of theovum is determined to be male if decolorization of DPPH occurs aftermore than about 1 hour. In another embodiment, the gender specificcompatibility of the ovum is determined to be male if decolorization ofDPPH occurs within about 15 minutes, whereas the gender specificcompatibility of the ovum is determined to be female if decolorizationof DPPH occurs from about 1 hour to about 24 hours.

The free radical scavenging activity may be measured by methylene blue.In one embodiment, the gender specific compatibility of the ovum isdetermined to be female if there is substantially no decolorization ofmethylene blue, whereas the gender specific compatibility of the ovum isdetermined to be male if there is decolorization of methylene bluewithin about 1 hour.

The present invention further provides a method of determining genderspecific compatibility of an ovum released in a menstrual cycle forpre-conception baby gender planning comprising the steps of: (a)contacting a body fluid from a non-pregnant female with a solid surfacehaving antibodies specific to estrogens, antibodies specific totestosterone, antibodies specific to progesterone and antibodiesspecific to hCG-beta; (b) contacting the solid surface from step (a)with at least one redox indicator; (c) measuring redox activity of thesolid surface; (d) comparing the redox activity of the solid surfacewith at least one standard; and (e) determining the gender specificcompatibility of the ovum released in the menstrual cycle. The redoxactivity may be measured by Ferric reducing/antioxidant power (FRAP)assay or Ferric reducing/antioxidant power using ferrictripyridyltriazine (FRAP-TPTZ) assay.

The present invention further provides a method for determining thegender of a fetus that is within a female's uterus using remotedetection devices. In vivo, a male fetus as a tissue mass has a higheroverall oxidizing activity, or a lower reducing activity, than thesurrounding maternal tissues, while a female fetus has a higher overallreducing or antioxidant activity than the surrounding maternal tissues.As is the case with post-conception or pre-conception urine tests, theredox activity of the fetal tissue or maternal tissue may be comparedwith a standard where the fetal gender is known. The remote detectiondevice of the present invention may comprise of a probe. The device maydetect the redox activity of different parts of a mother's internaltissues by non-invasively sensing the internal electrochemical signals,electromagnetic signals, or any other suitable physical and/or chemicalsignals. In one embodiment, internal redox potentials are detected bythe remote detection device. In another embodiment, internal electroniccharges are detected by the remote detection device. In a furtherembodiment, the remote detection device comprises an electroscope todetect electrochemical potential of fetal and maternal tissues.

The present remote detection device may comprise broadband diffuseoptical spectroscopy. (Lee et al. Noninvasive in vivo monitoring ofmethemoglobin formation and reduction with broadband diffuse opticalspectroscopy. J. Appl. Physiology 100: 615-622.) As known in the art,oxidation of proteins and amino acids generates ultra weak photonemission. The present remote detection device may comprise a sensitivephotomultiplier system to detect the ultra weak photon emission that isassociated with a male or female fetus. The present remote detectiondevice may comprise electron paramagnetic resonance imaging (EPRI) byusing redox sensitive paramagnetic contrast agents. (Yamada et el.Feasibility and assessment of non-invasive in vivo redox status usingelectron paramagnetic resonance imaging (EPRI). Actd Radiol. 43(4):433-40 (2002).) The present remote detection device may comprisemeasuring the surface oxygen tension of organs. (Miller A. T. Jr.University of North Carolina at Chapel Hill. Tissue oxygen tension andthe intracellular oxidation-reduction state. Personal communication.)Other suitable means may also be used for the present remote detectiondevice. See, Wang. Remote electrochemical sensors for monitoring organicand inorganic pollutants. Trends in Anal. Chem. 16: 84 (1997). Balcom etal. Spatial and temporal visualization of two aqueous ironoxidation-reduction reactions by nuclear magnetic resonance imaging. J.Chem Soc. Chem. Comm. 1992: 312-313. Livesey et al. Measurement oftissue oxidation-reduction state with carbon-13 nuclear magneticresonance spectroscopy. Cancer Research. 49: 1937-1940 (1989).

The change of the redox indicator may be analyzed with a scientificinstrument, such as spectrophotometer, fluorometer, turbiditimeter,luminometer, fluorescence meter or colorimeter. The change in color ofthe chromogenic molecule may also be observed visually. The change ofthe redox indicator may be converted to digital signals which can beprocessed by digital, electronic or other sensing scientific instrumentsor materials. The scientific instrument may convert the assay results toa digital text response using any suitable means such as an electronicprogramming In one embodiment, the instrument shows the text “It's aBOY” when the assay results suggest that the body fluid is from a femalecarrying a male fetus, and “It's a GIRL” when the assay results suggestthat the body fluid is from a female carrying a female fetus. Similarly,for a pre-conception assay on the body fluid from a non-pregnant female,the instrument may show “Time for a baby boy” if the ovum produced in aparticular menstrual cycle is compatible for fertilization by a Ychromosome-bearing sperm, and “Time for a baby girl” if the ovumproduced in a particular menstrual cycle is compatible for fertilizationby an X chromosome-bearing sperm.

The overall redox activity of the body fluid may be assayed using anelectrochemical sensor, which is able to produce an electrochemicallydetectable signal upon being reduced or oxidized. In this case, adetection device may be used which comprises the redox indictor and hasa disposable electrochemical cell, such as a thin layer electrochemicalcell. The sensing electrode may consist of platinum, palladium, carbon,indium oxide, gold, iridium, copper, steel or mixture thereof. U.S. Pat.No. 6,638,415. WO2002/006806. Carbon nanotubes may be used forelectrochemical sensing. Carbon nanotubes have the ability to promoteelectron transfer reactions when used as an electrode material inelectrochemical reactions. U.S. Patent Publication No. 20060096870.Electrochemistry and electrogenerated chemiluminescence with a singlefaradaic electrode may be used for the present methods. It comprises ofa faradaic working electrode and a capacitive counter electrode. Whenelectrical energy is supplied in the presence of a body fluid, afaradaic charge transfer occurs wherein at least one of (i) light, (ii)current. (iii) voltage and (iv) charge can be measured to determine thepresence or amount of analyte in a sample. WO2007/053191.

The steps of the methods of the present invention may be conducted by ahuman and/or a machine.

Encompassed by the present invention is a device configured to assay thebody fluid, process the data (i.e., the assay results), and present theresults (i.e., fetal gender related characteristics, for example, genderof the unborn child or biochemical environment of the uterus). Thedevice may include pre-installed software (for user control, and outputof information, as well as data processing/algorithmic data analysisetc.). The software may be designed based on the formulae describedabove. The device may contain at least one component to detectfluorescence, luminescence and/or other signal from within a chamber ofthe processing and/or sensing component.

In certain embodiments, blood may be added to a patch loaded with redoxchemicals or radical scavenging chemicals as described herein toevaluate fetal gender related characteristics. For example, non-invasivetransdermal membrane based diagnostic technology may be used accordingto the present invention. Additionally, special optics in electronicmeter can detect the color change and provide the evaluation. (U.S. Pat.No. 6,503,198.)

The overall redox activity of the body fluid may be assayed using anysuitable chemical, physical, biochemical or biological means. Theoverall redox activity of the body fluid may be assayed by contactingthe body fluid with at least one microorganism such as a bacterium, andobserving the physiological responses such as generation of coloredproduct, movement of the bacterium's cilia. The overall redox activityof the body fluid may be assayed by redox-sensitive green fluorescentprotein (GFP). Dooley et al. Imaging dynamic redox changes in mammaliancells with GFP indicators. J. Biol. Chem. 279, 24: 22284-22293 (2004).The overall redox activity of the body fluid may be measured by assayingfluorescence. In one embodiment, 5-cyano 2-3 ditolyl tetrazoliumchloride (CTC) is used for the present assay. CTC's oxidized form, whichis colorless and non-fluorescent, is readily reduced to fluorescent,insoluble CTC-formazan form.

The present methods may be modified to allow many samples to be testedand results read simultaneously using, for example, a microplate ormultiplex microarray. Additionally, the present methods may be modifiedto allow multiple sex hormones in a sample to be tested and results readsimultaneously using modern multiplex technology.

The present invention also provides an article of manufacture such as akit comprising (a) a urine collecting vial; (b) a solid substratecomprising a redox indicator, radicals (or a radical-generating system)or radical-sensitive agents, wherein the redox indicator comprises atleast one chromogenic chemical; (c) printed material instructing a womanto collect urine and contact the urine with the solid substrate; and (d)printed material instructing a woman to compare redox activity of theurine sample with at least one standard to determine the gender of theunborn child based on change in color in the chromogenic chemical. Thekit may also comprise other chemicals for processing and/or assaying theoverall redox activity of the body fluid. The printed matter may alsoindicate that, to determine the gender of an unborn child, the enclosedchemicals are to be used to assay the overall redox activity of the bodyfluid which will be compared with a standard. The printed material mayinstruct processing the urine prior to contact the urine with the solidsubstrate. The printed matter provides instructions as to how to use theenclosed chemicals for the test. Pictorial depictions of theinstructions may be included in the printed matter. The printed mattermay indicate that the body fluid is to be collected at any time during apregnancy, starting from the first day of missed menstruation. Theprinted matter may also indicate that optimum results may be obtained ifthe body fluid is collected between the 5th and 15th week of pregnancy.The printed matter may include color standards for resultinterpretations and indicates the color (or a range of colors) thatcorrelates with a male (or female) fetus. The chemicals may be providedin pre-measured quantities and may be in the form of solution, solidcrystals or dry film. The chemicals may be placed in capped vials orbottles and placed in separate compartments. The chemicals may beabsorbed or retained to solid supports and arranged sequentially to forma trickling column so as to ensure that various processes are conductedin a desired order. A kit may contain chemicals in sufficient amountsfor one, two or more assays.

Similarly, the present invention further provides an article ofmanufacture such as a kit comprising necessary chemicals for processingand/or assaying the overall redox activity or the radical scavengingactivity of the body fluid such as urine, as well as printed matterindicating that, to determine the suitable menstrual cycle forconceiving a baby of desired gender, the enclosed chemicals are to beused to assay the overall redox activity of the body fluid in at leasttwo consecutive menstrual cycles. The printed matter may indicate thatthe body fluid may be collected at any time during a menstrual cycle.The printed matter may also indicate that optimum results may beobtained if the body fluid is collected around the ovulation period. Theprinted matter provides instructions as to how to use the enclosedchemicals for the test. The printed matter also includes color standardsfor result interpretations and indicates the color (or a range ofcolors) that correlates with an ovum compatible with fertilization by aY chromosome-bearing sperm or an ovum compatible with fertilization byan X chromosome-bearing sperm. The printed matter indicates that twotypes of ova are produced in alternate months, and suggests that a womanwho wishes to have a baby boy (or girl) try to conceive in alternatemonths, starting from the month an ovum compatible with fertilization bya Y chromosome-bearing (or X chromosome-bearing) sperm is ovulated.

The redox activity may be measured on a solid substrate which comprisesa redox indicator, such as a chromogenic chemical, radicals (or aradical-generating system) or radical-sensitive agents. The chromogenicchemical may be impregnated on a strip. The article of manufacture ofthe present invention may be a test strip. The test strip may have asample application site where the body fluid may be applied. The bodyfluid may then pass through various aging, processing, filtering,adsorbing or chromatographic media (e.g., to allow selective onward flowof desired sex hormones) which ultimately leads to the procedure ofassaying the overall reducing or oxidizing activity and/or radicalscavenging capacity of the sample by reacting with at least one redoxindicator. The test strip may have more than one sample application siteso that the body fluid may be applied at more than one site on the stripat the same time. The same body fluid applied at different sites on thestrip may undergo different treatments and may meet differentchromogenic chemicals or redox indicators. The overall reducing oroxidizing activity of the sample is then evaluated based on a specificpattern of colors or other measurable signals.

In one embodiment, a strip and a digital strip reader are providedaccording to the present invention which would assay and calculate thelevels and/or the ratios of the sex hormones, and present the results indigital form.

Similarly, strips or kits containing various radical scavenging agents(such as methylene blue, DPPH or sodium salicylate) can be provided toevaluate radical scavenging capacity of a body fluid sample. Such stripsor kits may or may not contain parts to process the body fluid (e.g., toremove interfering compounds in the body fluid before having appropriatehormones react with radical scavenging agents). A change in color orproperties of the radical scavenging agents can then be used to evaluatefetal gender related characteristics.

An article of manufacture according to the present invention may containone type of assay method, or two or more suitable assay methods.

In specific examples, the accuracy of a body fluid sample having femalegender related characteristics can approach 100% if the processed sampleshows all of the following: (a) a transparent PTA test after 12 hr; (b)a DPPH test with discolorization within about 2 minutes; (c) a blue FRAPtest; and (d) a methylene blue test with no discolorization. Similarly,the accuracy of a body fluid sample having male gender relatedcharacteristics can approach 100% if the processed fluid shows all ofthe following a) a colloidal PTA test after 12 hr; b) a DPPH test withno discolorization; c) a golden yellow FRAP test; and (d) a methyleneblue test with discolorization within about 12 hours. These observationscan reverse depending upon sample processing technique and experimentalconditions.

Essentially all of the methods, articles of manufacture described hereinfor determining the gender of an unborn child after conception may alsobe used to determine gender specific compatibility of the ovum releasedin a menstrual cycle or for evaluation of uterine environment beforeconception for, for example, pre-conception baby gender planningDifferent methods may be used on the same sample to for increasedaccuracy of results.

The methods of the present methods may be used to test a body fluidobtained from any mammal, such as a human, horse, cow, dog, monkey,sheep, pig and cat.

The present invention also provides a method of conceiving a baby of adesired gender in a female by applying to the female a pharmaceuticalformulation with a specific sex hormone composition. Methods in theprior art are mostly based on modifying the pH value or ionic balance ofthe vaginal or uterine environment of a female, which have had limitedclinical success. How to Choose the Gender of Your baby, a book byAshley Spencer. Shettles et al., How to Choose the Sex of Your Baby.Broadway, 2006. The failure of those methods is largely due to thedetrimental effects that the pH-modifying (or ionic balance-modifying)composition has on the sperm motility and viability. In contrast, thepresent methods are more natural. As described above, the uterineenvironment and the changes that it undergoes during the normalmenstrual cycle can greatly influence gender selection. By takingadvantage of these naturally occurring characteristics, methods havebeen developed in the present invention to amplify this naturalselection process.

Specifically, as the hormonal composition of the uterus of a female (ora body fluid such as urine) prior to conception is associated with thecompatibility of the uterus to a male or female conception, the presentinvention provides for methods of increasing the chance of conceiving ababy of a desired gender in a female by mimicking (or amplifying) aspecific uterine environment. Therefore, by applying to the female apharmaceutical formulation comprising sex hormones of certaincomposition, the uterine environment of the female can be modified to becompatible with conception of a baby of the desired gender. Thepharmaceutical formulation may be applied, for example, near or aroundcervix uteri of the female. In certain embodiments, a pharmaceuticalformulation with relatively higher overall reducing activity wouldcreate a uterine environment (or amplify an existing uterineenvironment) to increase the chances of conceiving a female baby.Similarly, a pharmaceutical formulation with relatively higher overalloxidizing activity would create a uterine environment (or amplify anexisting uterine environment) to increase the chances of conceiving amale baby. It should be noted that in vivo conditions are similar to themild assaying conditions of the present invention, and that estrogensbehave as reducing agents whereas testosterone behaves as an oxidizingagent under in vivo conditions.

Table 2 tabulates an example of the pharmaceutical formulations that maybe applied to a female to conceive either a boy or a girl. Thepharmaceutical formulation may be in any suitable form, including, butnot limited to, a gel, a solution, a cream, a lotion, an ointment, afoam or a paste. For each type of formulation, the concentration rangesof the sex hormones, as well as the optimum concentrations, are shown.

TABLE 2 Concentration Optimum Hormone range (ng/ml) concentration(ng/ml) For Conception of A Boy Estradiol 1-35 14.6 ± 1.46 Estriol40-600 540 ± 54  Estrone 0.1-35  12.6 ± 1.26 Testosterone 1-25 13.6 ±1.36 Progesterone  1-500  192 ± 19.2 The concentrations the hormonesshould ensure that the [E − (T + P)]* value ranges from about 300 toabout 1,500. For Conception of A Girl Estradiol 1-20 11.7 ± 1.17 Estriol80-500  265 ± 26.5 Estrone 1-20  8.1 ± 0.81 Testosterone 5-80 27.2 ±2.72 Progesterone 100-1000  444 ± 44.4 The concentrations the hormonesshould ensure that the [E − (T + P)]* value ranges from about −10 toabout −800. *Although it should be the [E − (T + P + G)] value, as thecontribution of hCG before pregnancy is negligible, G can be omittedhere.

The hormones may be suspended in the pharmaceutical composition using anon-liquefying base containing purified water, and one or more offollowing components: propylene glycol, stearyl alcohol, white ceresinwax, mono- and di-glycerides, hypromellose 2208 (4000 cps), sodiumlauryl sulfate, methylparaben, edetate di-sodium andtertiary-butylhydroquinone. The pharmaceutical composition preferablycontains saline buffered at a physiological pH. The sex hormonecomposition of the pharmaceutical formulation is not limited to what istabulated in Table 2. The pharmaceutical formulation may contain sexhormones in certain concentrations to be in accordance with the specificranges taught in the present invention. For example, the formula(E+T)/(P+G) could be used to design pharmaceutical formulations for theconception of either a boy or a girl. Since contribution of hCG beforepregnancy is negligible, G can be omitted. Accordingly, to select for amale conception, the sex hormones would be in such concentrations togive an (E+T)/P value ranging from about 300 to about 1,500. Similarly,to select for a female conception, the sex hormones would be in suchconcentrations to give an (E+T)/P value ranging from about −10 to about−800.

Therefore, the present invention provides a method of conceiving a babyof a desired gender in a female comprising the step of applying to thefemale a pharmaceutical formulation selected from the group consistingof: (i) a pharmaceutical formulation comprising about 1 ng/ml to about35 ng/ml estradiol, about 40 ng/ml to about 600 ng/ml estriol, about 0.1ng/ml to about 35 ng/ml estrone, about 1 ng/ml to about 25 ng/mltestosterone, about 1 ng/ml to about 500 ng/ml progesterone when thedesired gender of the baby is male, wherein the [E−(T+P)] value rangesfrom about 300 to about 1,500, and (ii) a pharmaceutical formulationcomprising about 1 ng/ml to about 20 ng/ml estradiol, about 80 ng/ml toabout 500 ng/ml estriol, about 1 ng/ml to about 20 ng/ml estrone, about5 ng/ml to about 80 ng/ml testosterone, about 100 ng/ml to about 1000ng/ml progesterone when the desired gender of the baby is female,wherein the [E−(T+P)] value ranges from about −10 to about −800.

Sperm Sorting/Separation Based on Oxidizing/Reducing Environment

The composition of sex hormones and their ratios in two consecutivemenstrual cycles differ, as described elsewhere herein. One cycle hasmore testosterone and progesterone as compared to all the threeestrogens (estradiol, estrone and estriol) and this pattern reverses inthe next cycle, as described elsewhere herein. These cycle to cyclevariations impact the selective movement of sperm due to chemotacticforces, specifically oxidizing and reducing forces offered by individualhormones and signal transduction mechanisms inherent in sperm.

As described herein, the oxidizing/reducing properties of individualhormones control sperm selection. Hormones exhibit oxidizing or reducingproperties depending upon the environment. In vivo, testosterone andprogesterone show oxidizing properties whereas gonadotropins andestrogens show reducing properties. Out of these, hCG, which is onlypresent after pregnancy, always demonstrates reducing propertiesregardless of the environmental conditions. As described herein, theoverall oxidizing or reducing environment in the uterus, due to thesehormones, imparts ‘pulling’ and/or ‘pushing’ forces on the sperm. Insome embodiments, oxidizing conditions (higher T/E ratio) favor greateractivation and movement of Y chromosome containing sperm, resulting inconception of a male fetus, and reducing conditions (lower T/E ratio)favor activation and movement of X chromosome containing sperm,resulting in conception of a female fetus. In some embodiments, one ormore chemoattractants in the follicular fluid include progesterone,atrial natriuretic peptide, one or more odorants, bourgeonal, and/or thelike.

In some embodiments, the oxidizing or reducing environment is dependenton a composition of the follicular fluid released in the uterus.Described herein are methods for sorting/separating X and Y chromosomecontaining sperm while avoiding potentially harmful side-effects as aresult of typical sorting processes that use external forces tosort/separate sperm.

A method for sperm sorting includes: determining whether a female hasregular menstrual cycles; recovering follicular fluid from twoconsecutive cycles; freezing the follicular fluid until use; acquiringmid-cycle urine samples for the two consecutive cycles in whichfollicular fluid was taken; and assaying the two urine samples todetermine an oxidizing or reducing environment, as described elsewhereherein.

In some embodiments, the method further includes: providing amicrofluidics channel; collecting a plurality of sperm from a donor;differentially dyeing or labeling the plurality of sperm based on thepresence of either an X or a Y chromosome; applying the sperm to acentral region of the microfluidics channel; applying diluted follicularfluid from a first cycle to a first end of the microfluidics channel;applying diluted follicular fluid from a second cycle to a second end ofthe microfluidics channel; and assessing movement of the plurality ofsperm to the first and/or second end. Such movement may be recordedusing fluorescence microscopy and/or time lapsed videography. Asdescribed herein, Y-containing sperm move towards the follicular fluidrepresenting an oxidizing environment, while X-containing sperm movetowards the follicular fluid representing a reducing environment.

Intrauterine Insemination Based on Oxidizing/Reducing Environment

A method for intrauterine or intracervical insemination includes:determining a gender specific compatibility of an ovum released in amenstrual cycle of a non-pregnant female; determining whether thefallopian tubes of the non-pregnant female are unobstructed; collectingsemen from a male donor; cleaning a cervical area of the non-pregnantfemale; and when the fallopian tubes are unobstructed, dispensing thesemen in the cervix or in the uterine cavity using a flexible elongatemember. The method functions to allow the uterus time to select X or Ychromosome containing sperm depending upon the redox environment in theuterus.

In some embodiments, dispensing the semen in the cervix or uterinecavity is performed 1-3 times, at least three times, greater than threetimes, or substantially three times. For example, the inseminations aredone at least three times in a cycle starting on the thirteenth,seventeenth, and twentieth day after the start of menstrual cycle.

In some embodiments, collecting the semen from the male donor occursafter 1-2, 2-5, or more than five days of abstinence from ejaculation.

In some embodiments, the method includes washing a plurality of sperm inthe semen and concentrating the plurality of sperm in a small volume ofmedia. In some embodiments, the media comprises phosphate bufferedsaline or a physiological saline buffer. In some embodiments, the volumeis less than 1 mL, less than 0.5 mL, less than 0.25 mL, less than 0.1mL. In some embodiments, the volume is 0.1-0.5 mL, 0.5-1 mL, or anyrange or subrange therebetween.

In some embodiments, cleaning the cervical area includes positioning aspeculum in a vagina of the non-pregnant female.

In some embodiments, the elongate member includes a catheter.

The Examples illustrate embodiments of the invention and are not to beregarded as limiting.

Example 1 Post-Conception Tests Based on PTA Assay and FRAP Assay

Table 3 tabulates the results for post-conception tests conducted for4,524 women during the period between 1999 and 2008, with an averageobserved accuracy of about 90%.

At the initial stage, i.e., from the years 1999 through mid-2003, urinesamples were tested using sodium salicylate as the redox indicatorcombined with spectrophotometry (see Example 4). PITA test (see Example4) was started in 2002. FRAP assay (see Example 5) has been conductedsince 2008.

TABLE 3 Stage of pregnancy Boys Boys Girls Girls Accuracy for Accuracyfor YEAR (wks) Observed Reported Observed Reported Boys % Girls %1999-2001 7 212 242 229 258 87.6 88.8 8 305 344 331 367 88.7 90.2 9 299338 332 378 88.5 87.8 10 & up 281 328 293 345 85.4 84.9 2002 6 22 29 2325 75.9 92.0 7 21 31 18 19 67.7 94.7 8 32 36 19 21 88.9 90.5 9 17 21 2022 81.0 90.9 10 & up 46 54 26 31 85.2 83.9 2003 6 14 16 26 31 77.8 83.97 26 33 16 18 78.8 88.9 8 25 25 22 24 100.0 91.7 9 16 16 13 13 100.0100.0 10 & up 37 45 20 21 82.2 95.2 2004 6 16 19 13 15 84.2 86.7 7 26 3519 22 74.3 86.4 8 22 29 22 24 75.9 91.7 9 9 12 12 14 75.0 85.7 10 & up16 21 26 31 76.2 83.9 2005 5 10 11 5 5 90.9 100.0 6 13 14 13 14 92.992.9 7 21 26 20 24 80.8 83.3 8 8 9 9 10 88.9 90.0 9 9 10 12 13 90.0 92.310 & up 48 58 48 51 82.8 94.1 2006 5 22 23 20 22 95.7 90.9 6 30 33 24 2590.9 96.0 7 13 13 15 15 100.0 100.0 8 15 15 11 13 100.0 84.6 9 8 8 8 9100.0 88.9 10 & up 26 28 39 41 92.9 95.1 2007 5 19 21 32 36 90.5 88.9 616 18 13 15 88.9 86.7 7 16 18 17 18 88.9 94.4 8 20 24 15 15 83.3 100.0 912 13 12 14 92.3 85.7 10 & up 39 42 45 48 92.9 93.8 2008 5 26 28 34 3792.9 91.9 6 25 25 39 42 100.0 92.9 7 22 23 30 32 95.7 93.8 8 23 25 21 2292.0 95.5 9 28 30 25 25 93.3 100.0 10 & up 47 49 55 58 95.9 94.8 TOTAL1,958 2,241 2,042 2,283 88% 91.3% Total Fetal Gender 4,524 AverageObserved Predicted Accuracy: 90%

From 1999 to 2001 we tested urine samples collected after 21 days pastmissed menstruation date. Since 2005, we have been testing urine samplescollected as early as 1 day after missed menstruation, i.e., the 5thweek of pregnancy.

If the urine samples were aged for a longer period of time, the accuracyof the present methods could approach 100%. However, from a clinicalperspective, aging the urine samples for a long period of time is notapplicable. Accordingly, techniques to accelerate the aging process byphysical, chemical or biochemical means were developed.

Example 2 Post-Conception Tests Based on Calculation of (E+T)/(P+G)

The levels of the sex hormones were measured using commerciallyavailable ELISA kits (e.g., from Calbiotech Inc.).

Table 4 tabulates the results for post-conception tests conducted for 11women based on calculating the value of (E+T)/(P+G), showing thedifferent value ranges between women carrying a male fetus and womencarrying a female fetus. For women carrying a male fetus, the ratio of(E+T)/(P+G) is greater than about 1.4. For women carrying a femalefetus, the ratio of (E+T)/(P+G) is less than about 1.2.

TABLE 4 Sample # 1 2 3 4 5 6 14 15 16 17 18 Gender M M M M M M F F F F Fng/ml 4.4 6.1 14.4 21.9 11.6 22.9 19.6 6.8 8.0 68.0 33.6 ng/ml 1.0 3.51.3 20.9 28.8 32.1 20.4 4.4 8.5 10.4 14.9 ng/ml 42.2 167.7 388.0 1919.8310.5 426.8 444.7 97.4 330.6 104.5 348.6 ng/ml 0.3 4.5 1.0 19.8 34.715.5 16.0 1.0 1.7 10.0 11.8 ng/ml 11.3 109.0 78.3 444.1 176.2 333.8518.9 218.4 227.0 151.5 1106.3 mIU/ml 0.0 20.4 0.0 0.5 6.2 0.0 0.0 6.3250.0 3.2 0.0 27.7 40.3 297.6 1494.0 180.1 117.7 −57.4 −128.7 −144.1−97.8 −764.7 Oxi- Oxi- Oxi- Oxi- Oxi- Oxi- Re- Re- Re- Re- Re- dizingdizing dizing dizing dizing dizing ducing ducing ducing ducing ducingYellow Yellow Yellow Yellow Yellow Yellow Deep Blue Deep Blue Deep BlueDeep Blue Deep Blue Col- Col- Col- Col- Col- Col- Trans- Trans- Trans-Trans- Trans- loidal loidal loidal loidal loidal loidal parent parentparent parent parent 4.2 1.4 5.2 4.5 2.1 1.5 1.0 0.5 0.7 1.2 0.4 3.150.76

The “Oxidizing minus Reducing” value is calculated as(Estradiol+Estriol+Estrone)−(Testosterone+Progesterone+hCG). The“Overall Redox” is oxidizing if “Oxidizing minus Reducing” value ispositive; it is reducing if “Oxidizing minus Reducing” value isnegative.

Example 3 Post-Conception Tests Based on the HORAC Value or Calculationof HORAC/(E/T)

The levels of the sex hormones were measured using commerciallyavailable ELISA kits (e.g., Calbiotech). The HORAC value was measuredusing HORAC Assay Kit from Oxford Biomedical Research.

Table 5 tabulates the results for post-conception tests conducted for 26women based on the HORAC value or the value of HORAC/(E/T), showing thedifferent value ranges between women carrying a male fetus and womencarrying a female fetus.

TABLE 5 Testosterone Estradiol Estriol Estrone HORAC HORAC/HORAC/((Total E)/T) Sample # Gender ng/ml ng/ml ng/ml ng/ml HORAC Avg.((Total E)/T) AVG. 1 M 4.44 1.01 42.16 0.32 2000 204 2 M 6.14 3.54167.70 4.52 3000 105 3 M 14.45 1.32 388.04 0.95 2000 74 4 M 21.87 20.931919.75 19.85 4667 52 5 M 11.62 28.81 310.53 34.74 3995 124 6 M 22.9332.13 426.79 15.48 2000 97 7 M 2.12 22.50 555.56 17.32 3000 11 8 M 0.9024.66 536.74 14.48 3995 6 9 M 13.06 22.28 2009.26 14.17 2000 13 10 M4.81 34.72 424.64 55.89 8000 75 11 M 2.69 4.24 146.17 7.31 3995 68 12 M4.32 15.75 956.79 45.93 2000 8 27 M 61.74 21.81 436.12 13.23 1000 3204131 74 13 F 16.67 21.08 473.45 14.36 1000 33 14 F 19.64 20.39 444.7416.04 2000 82 15 F 6.77 4.41 97.37 1.04 8000 527 16 F 7.96 8.53 330.621.74 8000 187 17 F 68.03 10.35 104.55 10.02 7987 4349 18 F 33.60 14.87348.56 11.77 8000 716 19 F 17.69 22.75 332.06 12.36 6667 321 20 F 13.8020.20 216.51 18.13 7333 397 21 F 23.86 15.09 464.11 5.52 8000 394 22 F49.49 27.49 1469.14 52.15 8000 256 23 F 13.33 12.54 433.97 11.99 8000233 24 F 7.98 99.09 3486.73 172.31 7333 16 30 F 9.53 2.12 232.30 7.626000 6640 236 596

Except for three samples which had relatively high or low HORAC values(e.g., sample No. 10: 8000; sample No. 13: 1000; and sample No. 14:2000), the majority of the samples from women carrying a male fetus hadHORAC values less than about 5000; the majority of the samples fromwomen carrying a female fetus had HORAC values greater than about 6000.

In general, for women carrying a male fetus, the ratio of HORAC/(E/T) isless than about 150. For women carrying a female fetus, the ratio ofHORAC/(E/T) is greater than about 200.

Some values were out of the ranges of the majority of the samples, whichcannot be explained at this time. For example, sample No. 17 hadabnormally high value of HORAC/((Total E)/T) (4349). However, themajority of the samples had values falling within the ranges; thesamples between women carrying a male fetus and women carrying a femalefetus were statistically different. Accordingly, customers weregenerally asked to test their sample again after about a week, and, ifneeded, a third test would be recommended to achieve an improvedaccuracy.

Similarly, other formulae like HORAC/(E−(T+P+G)) and HORAC/(E+T)/(P+G)can also be calculated from the same data provided numbers for P and Gare known.

Example 4 Processing of Urine Samples for the PTA Assay

For the PTA assay, the urine samples were collected and processed asfollows. Post-conception urine samples were collected from about 2,782females at various time points of a pregnancy ranging from 4 days beforemissed menstruation to the 20th week of pregnancy. The customers wereshipped a vial for urine sample collection with a boric acid tablet inthe vial as a preservative. Customers were asked to collect urine voidedduring a resting period including early morning for at least two days.Customers who were working at night were asked to collect urine duringday-time instead for three consecutive resting periods. The samples werereturned to the clinic by mail which took from 2 to 5 days dependingupon the distance. The delivery of the urine sample by mail provided atime period for sample aging. The urine sample may be further aged atroom temperature for longer periods of time.

The urine was then processed as follows. 2 ml of the urine sample waspassed through acidic alumina to eliminate significant amount ofpigments and glucose. A C18 column was activated by methanol andpre-equilibrated with water. The cleared urine sample was then loaded onthe C18 column to be subjected to solid phase extraction (SPE). Afterthe urine sample passed through the C18 column, about 95% of sexhormones were retained on the column. The fraction of the urine samplethat passed through or did not bind to the column was referred to asFraction A. Fraction A was then treated with 50 ul of 10% Na2HPO4 and anexcess amount of calcium phosphate (about 160 mg) to remove bilirubin,urobilinogen, porphyrins, and other pigments. 50 ul of saturatedmercuric chloride was added to Fraction A to precipitate uric acid,ascorbic acid, creatine and some interfering proteins. 50 ul of 10% leadacetate solution was then added to Fraction A to remove creatine, someinterfering proteins and bilirubin. 10 mg of barium chloride crystalswas added to Fraction A to remove bilirubin, remaining interferingproteins and some interfering inorganic oxidants/reductants. The pH ofFraction A was then adjusted to 8.5 with 50 ul to 70 ul of 1 M sodiumhydroxide, followed by centrifugation or filtration to remove theinsoluble components.

In the PTA test, 20 mg of crystalline phosphotungstic acid was added to0.75 ml of Fraction A, and the solution was kept below 21° C. for 12hours. If the solution turned to a stable white fluorescent colloidalsuspension after 12 hours, the urine sample was reported to be from afemale exhibiting male gender related characteristics. If the solutioninitially formed white precipitates which later disappeared to result ina transparent solution, the urine sample was reported to be from afemale exhibiting female gender related characteristics. Repeatedanalysis of the same urine sample aged for different periods of timeimproved the accuracy of the analytical results.

Example 5 Processing of Urine Samples for a FRAP Assay

For the FRAP assay, the urine samples were processed as follows. Theurine samples were treated as above in Example 2; however, in this casethe components that were retained on the C18 column were eluted andtested.

2 ml of the urine sample was passed through acidic alumina to eliminatesignificant amount of pigments and glucose. A C18 column was activatedby methanol and pre-equilibrated with water. The cleared urine samplewas then loaded on the C18 column After the urine sample passed throughthe C18 column, about 95% of sex hormones were retained on the column,which were than eluted with 1 ml of HPLC grade methanol and calledFraction B. Fraction B was diluted with 2 ml water. 2.5 ml of dilutedFraction B was mixed with 2.5 ml of 0.2 M phosphate buffer (pH 6.6) and2.5 ml of 1% potassium ferricyanide. The mixture was incubated at 50° C.for 20 min and cooled to room temperature. The mixture was then mixedwith 2.5 ml of 10% trichloroacetic acid and centrifuged at 6500 rpm for10 min. The supernatant (2.5 ml) was mixed with 2.5 ml distilled water,before 0.5 ml of 0.1% ferric chloride was added. In this assay, theresults were read within one minute after the addition of the ferricchloride, as the results were not stable at later time points.

If Fraction B turned to deep blue after the FRAP assay, the urine samplewas reported to be exhibiting male gender related characteristics. IfFraction B turned to golden yellow, faint yellow or faint green afterthe FRAP assay, the urine sample was reported to be exhibiting femalegender related characteristics. Repeated analysis of the same urinesample aged for different periods of time improved the accuracy of theanalytical results.

If the same urine sample was analyzed by more than one type of assay, wehave found that the accuracy of the results can approach 100%. Forexample, if the PTA test on Fraction A produced a stable whitefluorescent colloidal suspension after 12 hrs, and the 1-RAP test onFraction B resulted in a deep blue solution, it was almost 100% accuratethat the urine sample was exhibiting male gender relatedcharacteristics. Similarly, if the PTA test on Fraction A produced atransparent solution after 12 hrs, and the FRAP test on Fraction Bresulted in a solution with golden yellow, faint yellow or faint greenin color, the accuracy was close to 100% that the urine sample wasexhibiting female gender related characteristics.

Alternatively, the FRAP test can also be performed on Fraction A. Whenurine samples were aged for at least 3 months at room temperature, theaccuracy of this method conducted was close to 100%.

Example 6 Redox Activity Assay with Sodium Salicylate

The urine samples were processed and assayed using sodium salicylate asfollows. For post-conception urine sample assays, customers were askedto collect early morning urine samples on three consecutive mornings anddrop them off at the clinic. All three samples from the same customerwere studied separately and a report was issued after pooling theresults. To process the urine sample, 4 ml of urine sample was treatedwith 20 mg of lead acetate for 30 minutes to precipitate interferingproteins. 1 ml of processed urine sample and 1 ml of 0.75 M hydrochloricacid were added to a 10 ml glass vial, before 1 ml of 1% sodiumsalicylate was added slowly to the vial which was gently shaken to mixthe reagents. Controls for the test were also included where, instead of1 ml of 1% sodium salicylate, 1 ml of distilled water was added to thevial. The reaction mixture was incubated at 50° C. for 2 hours and thencooled to room temperature. The absorbance of the reaction mixture atwavelengths between 470 nm to 550 nm was then measured against controlin a spectrophotometer. When a peak was observed around wavelength 510nm, a result of a male fetus was recorded for this sample. When a troughor a straight base line was observed around wavelength 510 nm, a resultof a female fetus was recorded for this sample.

Example 7 FRAP Assay with Fe(III)-TPTZ

Fe(III)-TPTZ may also be used as the redox indicator for the FRAP assay.Under acidic conditions, Fe(III)-TPTZ complex can be reduced toFe(II)-TPTZ. Fe(II)-TPTZ has intense blue color and can be monitored atwavelength 593 nm. FRAP working solution was freshly prepared by mixing10 ml 300 mmol/1 acetate buffer (pH 3.6), 1.0 ml 10 mmol/1 TPTZ (2,4,6tripyridyl-s-triazine) in 40 mmo1/1HCl solution, and 1.0 ml 20mmol/FeCl₃.6H₂O solution.

2 drops of processed urine sample were added to 0.5 ml FRAP workingsolution and the solution was incubated for 10 minutes. Under the harshconditions of the FRAP assay, the urine from a female exhibiting malegender related characteristics has a comparatively higher overallreducing activity than urine from a female exhibiting female genderrelated characteristics. Accordingly, a urine sample from a femalecarrying a male fetus was able to reduce Fe(III)-TPTZ to Fe(II)-TPTZ,with the reaction mixture turned dark blue. In contrast, a urine samplefrom a female carrying a female fetus was not able to reduceFe(III)-TPTZ, and thus, the reaction mixture remained yellow. When urinesamples were aged for at least 3 months at room temperature, theaccuracy of this method conducted on 161 urine samples was close to100%. Among the 161 urine samples, 67 samples were from females carryinga male fetus; 94 samples were from females carrying a female fetus.

Example 8 Redox Activity Assay with Polyaniline-Coated Film

Polyaniline-coated film was prepared by chemical deposition ofpolyaniline on a transparent film of poly (ethylene terephthalate). 100ml of the reaction mixture comprised 1 M sulfuric acid, 1 ml of aniline,0.9 g of potassium iodate and 1 g of 5-sulfosalicyclic acid. U.S. Pat.No. 5,451,526. A deposition time of 2.5 hours at room temperature wasused to produce the polyaniline film. The polyaniline coatings on bothsides of the film were carefully washed to remove loose deposits. Thefilm was dried, cut into small pieces (3 mm×2.0 cm), and stored in aclean container before use Immediately prior to use, the film wasequilibrated in an acidic ferric chloride solution for 2 minutes toconvert all the polyaniline to its emaraldine state, which is theoxidized state. 1.75 ml of urine was processed by passing through 160 mgof acidic alumina column, and transferred to a cuvette. ThePolyaniline-coated film was carefully washed with deionized water andtransferred to the cuvette containing aged or processed urine. Theoptical density of the liquid in the cuvette was read at wavelength 630nm on a spectrophotometer. Alternatively, after washing, the film wasdipped in the purified urine fraction for 1 second before being takenout for visual observation of its change in color. Under the mildcondition of this assay, the aged and processed urine from a femalecarrying a female fetus has a comparatively higher overall reducingactivity than urine from a female carrying a male fetus, and therefore,was able to reduce the emeraldine form of polyaniline (blue) to its morereduced leucoemeraldine state (off-white). An aged and processed urinesample from a female bearing a male fetus did not react with theemeraldine form of polyaniline (blue). As a result, the film remainedblue in color. When fresh urine samples were used without processing, nodifference could be observed in this test.

Example 9 Pre-Conception Assay

The overall redox activity of the body fluid during a menstrual cyclewithin which a female conceives is similar to the redox activity of herbody fluid after conception. In other words, if conception occurs whenmidcycle urine sample of non-pregnant woman has an overall higherreducing activity, the process of conception “locks” this condition anda similar higher reducing activity is exhibited by urine sample from thesame woman anytime after pregnancy. Namely, if her pre-conception bodyfluid has a comparatively higher overall reducing activity than that ofthe body fluid from a non-pregnant female carrying an ovum compatiblewith fertilization by an X chromosome-bearing sperm under the harshcondition (for example, in the FRAP assay), her ovum is compatible withfertilization by a Y chromosome-bearing sperm. Consistently, herpost-conception urine sample would also have a higher overall reducingactivity than that of urine from a female carrying a female fetus, whichis correlated with a male fetus. Conversely, if her pre-conception urinesample has a higher overall oxidizing activity than that of urine from anon-pregnant female carrying an ovum compatible with fertilization by aY chromosome-bearing sperm under harsh conditions, her ovum iscompatible with fertilization by an X chromosome-bearing sperm.Likewise, her post-conception urine sample would also have a higheroverall oxidizing activity than that of urine from a female carrying amale fetus, which is correlated with a female fetus.

The FRAP assay was used to test both pre-conception and post-conceptionurine samples obtained from the same customer. Pre-conception urinesamples were collected during ovulation period for at least twoconsecutive months. Post-conception urine samples were collected on thefirst or second day of missed menstruation due to pregnancy. If thepre-conception samples indicated an alternating ovulation pattern, thenthe customer were advised as follows. If the customer desired a babyboy, she was advised to try conception in months when the ova beingproduced were compatible with fertilization by a Y chromosome-bearingsperm. If the customer desired a baby girl, she was advised to tryconception in months when the ova being produced were compatible withfertilization by an X chromosome-bearing sperm (see Table 7 below).

Those customers whose urine samples indicated ova being released in twoconsecutive months had the same gender specific compatibilities wereadvised to collect more urine samples for additional tests. However,customers of this category did not wish to incur more costs and,therefore, did not continue after the initial tests.

Table 6 tabulates the results of pre-conception tests we conducted for130 customers during the period between 2006 and August 2008.

TABLE 6 Number of customers who received the pre-conception test andlater the post-conception test = 130 Number of customers Number ofcustomers Number of customers Number of customers who desired a baby whoconfirmed having who desired a baby who confirmed having boy and tried ababy boy = 52 girl and tried a baby girl = 41 conception according (Fewdid not call back*.) conception according (Few did not call back*.) toour method = 71 to our method = 59 Success rate for conception Successrate for conception of baby boy = 73.2% of baby girl = 69.5% Totalsuccess rate of pre-conception test = 71.3% *As a policy, we never calla customer for a feedback due to confidentiality and privacy concerns.

Example 10 Pre-Conception Test Based on FRAP Assay and PTA Assay

Table 7 tabulates the results of pre-conception tests we conducted for 8customers to assay the alternating ovulation pattern.

TABLE 7 SAMPLE ID DATE FRAP TEST 12 HR PTA TEST STATUS REPORTED K K May28, 2008 BLUE Negative Jun. 28, 2008 YELLOW Positive Jul. 28, 2008 NSAssumed Negative Aug. 28, 2008 NS Assumed Positive Missed Sep. 13, 2008Conception in Positive Tested Sep. 30, 2008 M YELLOW White ColloidalDELIVERED M D K October 2007 BLUE Negative November 2007 YELLOW PositiveDecember 2007 NS Assumed Negative January 2008 NS Assumed PositiveFebruary 2008 NS Assumed Negative March 2008 NS Assumed Positive April2008 NS Assumed Negative May 2008 NS Assumed Positive Conceived June2008 Conception in Positive Tested Jun. 26, 2008 M YELLOW WhiteColloidal DELIVERED M P D Aug. 3, 2008 BLUE Negative Sep. 12, 2008YELLOW Positive Missed Sep. 26, 2008 Conception in Positive Tested Oct.8, 2008 M YELLOW White Colloidal DELIVERED M J B Jun. 18, 2007 YELLOWPositive Jul. 15, 2007 BLUE Negative Aug. 12, 2007 NS Assumed PositiveSep. 10, 2007 NS Assumed Negative Oct. 7, 2007 NS Assumed PositiveMissed Oct. 25, 2007 Conception in Positive Tested Oct. 29, 2007 MYELLOW White Colloidal DELIVERED M S M Sep. 13, 2006 YELLOW PositiveOct. 10, 2006 BLUE Negative Missed Oct. 25, 2006 Conception in NegativeTested Nov. 17, 2006 BLUE Transparent DELIVERED F J R Aug. 22, 2008 BLUENegative Sep. 20, 2008 YELLOW Positive Oct. 18, 2008 NS Assumed NegativeNov. 16, 2008 NS Assumed Positive Dec. 14, 2008 NS Assumed NegativeMissed Dec. 28, 2008 Conception in Negative Tested Jan. 12, 2008 BLUETransparent DELIVERED F R G Jun. 8, 2006 FAINT BLUE* Negative Jul. 5,2006 YELLOW Positive Aug. 4, 2006 NS Assumed Negative Sep. 1, 2006 NSAssumed Positive Sep. 27, 2006 BLUE NEGATIVE Missed Oct. 10, 2006Conception in Negative Tested Nov. 12, 2006 BLUE Transparent DELIVERED FR T May 5, 2007 BLUE Negative Jun. 3, 2007 YELLOW Positive Jul. 1, 2007NS Assumed Negative Jul. 30, 2007 NS Assumed Positive Aug. 2, 2007 NSAssumed Negative Missed Aug. 16, 2007 Conception in Negative Tested Sep.3, 2007 YELLOW* Transparent REPORTED M, DELIVERED F All samples weretested +/−1 day near ovulation. NS = Not studied. *= Exception.

Pre-conception test was done for two consecutive months for every womanto assay the alternating pattern of ovulation. Almost 90% women hadalternating reproductive environment.

Example 11 Pre-Conception Test Based on the HORAC Value and the Ratio ofHORAC/(E/T)

Table 8 tabulates the results of pre-conception tests we conducted for 2customers showing the HORAC value and the ratio of HORAC/(E/T).

TABLE 8 Testosterone Estradiol Estriol Estrone HORAC/ HORAC/((TotalE)/T) Sample # Gender ng/ml ng/ml ng/ml ng/ml HORAC ((Total E)/T) AVG.27 M 7.13 1.56 117.46 0.39 2000 119 145 (1st pre-con test) 27 6.77 1.88116.03 0.60 3000 171 (2nd pre-con test) 30 F 5.39 0.65 57.17 1.42 3995364 694 (1st pre-con test) 30 16.72 6.50 126.08 3.41 8333 1025 (2ndpre-con test)

Similarly, other formulae like HORAC/(E−(T+P+G)) and HORAC/(E+T)/(P+G)can also be calculated from the same data above provided numbers for Pand G are also evaluated.

Examples 12-16 illustrate various methods to process urine samplesbefore assaying its overall redox activity.

Example 12 Processing of Urine Samples with Acidic Alumina, CalciumChloride and Lead Acetate

The pH of 3 ml urine sample was adjusted to 6.4 with 1 M HCl. The urinesample was then centrifuged to remove insoluble components. Thesupernatant was loaded to a column containing 200 mg acidic alumina toremove interfering agents. 50 ul of 10% Na₂HPO₄ and 80 mg of calciumchloride were then added to the cleared urine sample. The reactionmixture was incubated at room temperature for 5 minutes. 10 mg of leadacetate was added to the sample and the test tube was shaken until alllead acetate dissolved. After 5 minutes, the pH of the sample wasadjusted to 8.5 with 2.5 M NaOH. The sample was then centrifuged at12000 rpm for 2 minutes to remove insoluble components. The processedurine sample was then assayed using the PTA or FRAP assay (Examples 2and 3). The FRAP assay worked better at a basic pH here.

Example 13 Processing of Urine Samples with Sulfuric Acid and Florisil

1 ml of fresh urine was added to a microcentrifuge tube and incubated onice. Concentrated sulfuric acid was slowly added to the urine sample toreach its final concentration of 3 M in the urine sample. Themicrocentrifuge tube was inverted slowly several times and the mixtureincubated at 37° C. for 24 hours. The sample was then centrifuged at12000 rpm for 2 minutes before the supernatant was passed throughFlorisil to result in processed urine sample. 10 mg of crystallinephospho-24-tungstic acid was then added to 0.75 ml of this processedurine sample, and the PTA assay conducted (Example 2).

Example 14 Processing of Urine Samples with Ion Exchange Resins

2 ml of urine sample was adjusted to pH 5.3 with diluted glacial aceticacid. The urine sample was then centrifuged at 12000 rpm for 2 minutes.The supernatant was batch treated with 160 mg of well washed Dowex™resins (Dow Chemical Company) IX 8-100 mesh Cl⁻ form ion exchange resinto remove porphyrins. This cleared urine sample was then processed asdescribed in Example 8. The processed urine sample was assayed using thePTA or FRAP assay (Examples 2 and 3).

Example 15 Processing of Urine Samples with Nylon Filter

The pH of 2 ml of urine sample was adjusted to 4.0 with 1 M HCl, beforethe urine sample was centrifuged at 12000 rpm for 2 minutes. Thesupernatant was passed through 0.2 um Nylon filter to remove porphyrins.This cleared urine sample was then processed as described in Example 8.Example 16 Processing of Urine Samples with Acidic Alumina and Talc

Example 16 Processing of Urine Samples with Acidic Alumina and Talc

In order to develop a home based gender test, 2 ml of urine sample wasadded to 200 mg of acidic alumina and talc, and the tube was shaken toensure mixing. The sample was incubated at room temperature for 5minutes, before the sample was filtered to obtain processed urinesample. The processed urine sample was assayed using the FRAP assay(Example 3).

The scope of the present invention is not limited by what has beenspecifically shown and described hereinabove. Those skilled in the artwill recognize that there are suitable alternatives to the depictedexamples of materials, configurations, constructions and dimensions.Numerous references, including patents and various publications, arecited and discussed in the description of this invention. The citationand discussion of such references is provided merely to clarify thedescription of the present invention and is not an admission that anyreference is prior art to the invention described herein. All referencescited and discussed in this specification are incorporated herein byreference in their entirety. Variations, modifications and otherimplementations of what is described herein will occur to those ofordinary skill in the art without departing from the spirit and scope ofthe invention. While certain embodiments of the present invention havebeen shown and described, it will be obvious to those skilled in the artthat changes and modifications may be made without departing from thespirit and scope of the invention. The matter set forth in the foregoingdescription is offered by way of illustration only and not as alimitation.

What is claimed is:
 1. A method for intrauterine or intracervicalinsemination of a non-pregnant female, the method including: determininga gender specific compatibility of an ovum released in a menstrual cycleof the non-pregnant female, wherein a net reducing activity of themenstrual cycle indicates a female gender specific compatibility of theovum and a net oxidizing activity of the menstrual cycle indicates amale gender specific compatibility of the ovum; determining whether oneor both fallopian tubes of the non-pregnant female are unobstructed toallow release of the ovum to increase fertilization potential;collecting semen from a male donor; cleaning a cervical area of thenon-pregnant female; and when one or both fallopian tubes areunobstructed, dispensing the semen in a cervix or a uterine cavity ofthe non-pregnant female using a flexible elongate member, wherein an Xchromosome containing sperm subset of the semen traffics to the ovumwhen there is the net reducing activity of the menstrual cycle, andwherein a Y chromosome containing sperm subset of the semen traffics tothe ovum when there is the net oxidizing activity of the menstrualcycle.
 2. The method of claim 1, further comprising collecting a firstfollicular fluid sample from the non-pregnant female during a firstmenstrual cycle and collecting a second follicular fluid sample from thenon-pregnant female during a second menstrual cycle.
 3. The method ofclaim 2, wherein the first menstrual cycle is consecutive with thesecond menstrual cycle.
 4. The method of claim 2, further comprisingseparating the semen into X chromosome containing sperm and Y chromosomecontaining sperm by surrounding them with the first follicular fluidsample and the second follicular fluid sample, wherein the X chromosomecontaining sperm are attracted to the first follicular fluid sample andthe Y chromosome containing sperm are attracted to the second follicularfluid sample.
 5. The method of claim 4, wherein the X chromosomecontaining sperm and the Y chromosome sperm are differentially labeledwith a label.
 6. The method of claim 5, wherein the label comprises afluorophore.
 7. The method of claim 4, wherein dispensing the semencomprises only dispensing the X chromosome or Y chromosome containingsperm in the cervix or the uterine cavity of the non-pregnant female. 8.The method of claim 7, wherein the X chromosome containing sperm aredispensed when the gender specific compatibility of the ovum indicatesthe female gender, and the Y chromosome containing sperm are dispensedwhen the gender specific compatibility of the ovum indicates the malegender.
 9. The method of claim 2, wherein one or more synthetic hormonalformulations are used in place of one or more of the first and secondfollicular fluids.
 10. The method of claim 9, wherein the synthetichormonal formulation comprises a mixture of sex hormones where((E−(T+P)) is less than 1.0 to activate X chromosome containing sperm.11. The method of claim 9, wherein the synthetic hormonal formulationcomprises a mixture of sex hormones where ((E−(T+P)) is greater than 1.0to activate Y chromosome containing sperm.
 12. The method of claim 1,wherein dispensing the semen comprises dispensing the semen 2-4 times inthe menstrual cycle.
 13. The method of claim 12, wherein there is a timeperiod of 2-4 days between each dispensing.
 14. The method of claim 1,wherein collecting the semen from the male donor occurs after 2-5 daysof abstinence from ejaculation.
 15. The method of claim 1, furthercomprising washing a plurality of sperm in the semen and concentratingthe plurality of sperm in a volume of media.
 16. The method of claim 1,wherein cleaning the cervical area includes positioning a speculum in avagina of the non-pregnant female.
 17. The method of claim 1, whereinthe method is performed in an absence of ovum or follicle-inducingchemicals.
 18. The method of claim 1, determining the gender specificcompatibility of the ovum comprises: obtaining a urine sample fluid fromthe non-pregnant female; aging the urine sample at ambient temperaturefor at least about 2 days; passing the aged urine sample over a solidsurface comprising antibodies specific to an estrogen, testosterone,progesterone, and human chorionic gonadotropin beta subunit (hCG-beta);contacting the solid surface with at least one redox indicator;detecting a net redox activity of the estrogen, testosterone,progesterone, and hCG-beta retained on the solid surface by theantibodies, wherein the net redox activity is detected by a change in achemical property of the at least one redox indicator; and comparing thedetected net redox activity to a known net redox activity of at leastone standard.
 19. The method of claim 18, wherein the at least one redoxindicator is selected from the group consisting of: sodium salicylate,methylene blue, a heteropoly acid or its salt, a chromogenic chemical, afluorophore, a redox sensitive polymer, and combinations thereof. 20.The method of claim 18, further comprising processing the urine sampleprior to contacting the aged urine sample with the solid surface.